Chiral control

ABSTRACT

The present invention relates to chirally controlled oligonucleotides, chirally controlled oligonucleotide compositions, and the method of making and using the same. The invention specifically encompasses the identification of the source of certain problems with prior methodologies for preparing chiral oligonucleotides, including problems that prohibit preparation of fully chirally controlled compositions, particularly compositions comprising a plurality of oligonucleotide types. In some embodiments, the present invention provides chirally controlled oligonucleotide compositions. In some embodiments, the present invention provides methods of making chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Division of application Ser. No. 15/462,787, filedon Mar. 17, 2017, issued as U.S. Pat. No. 10,590,413, which is aDivision of application Ser. No. 14/414,614, filed on Jan. 13, 2015,issued as U.S. Pat. No. 9,982,257, which is a 371 National StageApplication of PCT International Application PCT/US13/50407, filed Jul.12, 2013, which claims priority to U.S. Provisional Application Ser. No.61/671,655, filed Jul. 13, 2012, 61/671,656, filed Jul. 13, 2012,61/671,722, filed Jul. 14, 2012, and 61/671,724, filed Jul. 14, 2012,the entirety of each of which is incorporated herein by reference.

SEQUENCE LISTING

In accordance with 37 CFR 1.52(e)(5), a Sequence Listing in the form ofa text file (entitled “SequenceListing.txt,” created on Aug. 19, 2013,and 531 kilobytes) is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Oligonucleotides are useful in therapeutic, diagnostic, research andnanomaterials applications. The use of naturally occurring nucleic acids(e.g., unmodified DNA or RNA) for therapeutics can be limited, forexample, because of their instability against extra- and intracellularnucleases and/or their poor cell penetration and distribution.Additionally, in vitro studies have shown that properties of antisenseoligonucleotides such as binding affinity, sequence specific binding tothe complementary RNA (Cosstick and Eckstein, 1985; LaPlanche et al.,1986; Latimer et al., 1989; Hacia et al., 1994; Mesmaeker et al., 1995),and stability to nucleases can be affected by the absolutestereochemical configurations of the phosphorus atoms (Cook, et al.US005599797A). Therefore, there is a need for new and improvedoligonucleotide compositions.

SUMMARY OF THE INVENTION

The present invention encompasses the recognition that there exists aneed for chirally controlled oligonucleotide compositions and newmethods for synthesizing the same. The invention specificallyencompasses the identification of the source of certain problems withprior methodologies for preparing chiral oligonucleotides, includingproblems that prohibit preparation of fully chirally controlledcompositions, particularly compositions comprising a plurality ofoligonucleotide types.

In some embodiments, the present invention provides chirally controlledoligonucleotide compositions.

In some embodiments, the present invention provides methods of makingchirally controlled oligonucleotides and chirally controlledoligonucleotide compositions.

In some embodiments, the present invention provides methods of usingchirally controlled oligonucleotide and chirally controlledoligonucleotide compositions.

All publications and patent documents cited in this application areincorporated herein by reference in their entirety.

Definitions

Aliphatic: The term “aliphatic” or “aliphatic group”, as used herein,means a straight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic or polycyclic hydrocarbon that is completely saturated orthat contains one or more units of unsaturation, but which is notaromatic (also referred to herein as “carbocycle” “cycloaliphatic” or“cycloalkyl”), that has a single point of attachment to the rest of themolecule. In some embodiments, aliphatic groups contain 1-50 aliphaticcarbon atoms. Unless otherwise specified, aliphatic groups contain 1-10aliphatic carbon atoms. In some embodiments, aliphatic groups contain1-6 aliphatic carbon atoms. In some embodiments, aliphatic groupscontain 1-5 aliphatic carbon atoms. In other embodiments, aliphaticgroups contain 1-4 aliphatic carbon atoms. In still other embodiments,aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet otherembodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. Insome embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”)refers to a monocyclic or bicyclic C₃-C₁₀ hydrocarbon that is completelysaturated or that contains one or more units of unsaturation, but whichis not aromatic, that has a single point of attachment to the rest ofthe molecule. In some embodiments, “cycloaliphatic” (or “carbocycle” or“cycloalkyl”) refers to a monocyclic C₃-C₆ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule. Suitable aliphatic groups include, but are notlimited to, linear or branched, substituted or unsubstituted alkyl,alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

Alkylene: The term “alkylene” refers to a bivalent alkyl group. An“alkylene chain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein nis a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3,from 1 to 2, or from 2 to 3. A substituted alkylene chain is apolymethylene group in which one or more methylene hydrogen atoms arereplaced with a substituent. Suitable substituents include thosedescribed below for a substituted aliphatic group.

Alkenylene: The term “alkenylene” refers to a bivalent alkenyl group. Asubstituted alkenylene chain is a polymethylene group containing atleast one double bond in which one or more hydrogen atoms are replacedwith a substituent. Suitable substituents include those described belowfor a substituted aliphatic group.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, and/or worms. In some embodiments, ananimal may be a transgenic animal, a genetically-engineered animal,and/or a clone.

Approximately: As used herein, the terms “approximately” or “about” inreference to a number are generally taken to include numbers that fallwithin a range of 5%, 10%, 15%, or 20% in either direction (greater thanor less than) of the number unless otherwise stated or otherwise evidentfrom the context (except where such number would be less than 0% orexceed 100% of a possible value). In some embodiments, use of the term“about” in reference to dosages means±5 mg/kg/day.

Aryl: The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic andbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains three to seven ring members. The term “aryl”may be used interchangeably with the term “aryl ring.” In certainembodiments of the present invention, “aryl” refers to an aromatic ringsystem which includes, but not limited to, phenyl, biphenyl, naphthyl,anthracyl and the like, which may bear one or more substituents. Alsoincluded within the scope of the term “aryl,” as it is used herein, is agroup in which an aromatic ring is fused to one or more non-aromaticrings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

Characteristic portion: As used herein, the phrase a “characteristicportion” of a protein or polypeptide is one that contains a continuousstretch of amino acids, or a collection of continuous stretches of aminoacids, that together are characteristic of a protein or polypeptide.Each such continuous stretch generally will contain at least two aminoacids. Furthermore, those of ordinary skill in the art will appreciatethat typically at least 5, 10, 15, 20 or more amino acids are requiredto be characteristic of a protein. In general, a characteristic portionis one that, in addition to the sequence identity specified above,shares at least one functional characteristic with the relevant intactprotein.

Characteristic sequence: A “characteristic sequence” is a sequence thatis found in all members of a family of polypeptides or nucleic acids,and therefore can be used by those of ordinary skill in the art todefine members of the family.

Characteristic structural element: The term “characteristic structuralelement” refers to a distinctive structural element (e.g., corestructure, collection of pendant moieties, sequence element, etc) thatis found in all members of a family of polypeptides, small molecules, ornucleic acids, and therefore can be used by those of ordinary skill inthe art to define members of the family.

Comparable: The term “comparable” is used herein to describe two (ormore) sets of conditions or circumstances that are sufficiently similarto one another to permit comparison of results obtained or phenomenaobserved. In some embodiments, comparable sets of conditions orcircumstances are characterized by a plurality of substantiallyidentical features and one or a small number of varied features. Thoseof ordinary skill in the art will appreciate that sets of conditions arecomparable to one another when characterized by a sufficient number andtype of substantially identical features to warrant a reasonableconclusion that differences in results obtained or phenomena observedunder the different sets of conditions or circumstances are caused by orindicative of the variation in those features that are varied.

Dosing regimen: As used herein, a “dosing regimen” or “therapeuticregimen” refers to a set of unit doses (typically more than one) thatare administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regimen, which may involve one or more doses. In someembodiments, a dosing regimen comprises a plurality of doses each ofwhich are separated from one another by a time period of the samelength; in some embodiments, a dosing regime comprises a plurality ofdoses and at least two different time periods separating individualdoses. In some embodiments, all doses within a dosing regimen are of thesame unit dose amount. In some embodiments, different doses within adosing regimen are of different amounts. In some embodiments, a dosingregimen comprises a first dose in a first dose amount, followed by oneor more additional doses in a second dose amount different from thefirst dose amount. In some embodiments, a dosing regimen comprises afirst dose in a first dose amount, followed by one or more additionaldoses in a second dose amount same as the first dose amount.

Equivalent agents: Those of ordinary skill in the art, reading thepresent disclosure, will appreciate that the scope of useful agents inthe context of the present invention is not limited to thosespecifically mentioned or exemplified herein. In particular, thoseskilled in the art will recognize that active agents typically have astructure that consists of a core and attached pendant moieties, andfurthermore will appreciate that simple variations of such core and/orpendant moieties may not significantly alter activity of the agent. Forexample, in some embodiments, substitution of one or more pendantmoieties with groups of comparable three-dimensional structure and/orchemical reactivity characteristics may generate a substituted compoundor portion equivalent to a parent reference compound or portion. In someembodiments, addition or removal of one or more pendant moieties maygenerate a substituted compound equivalent to a parent referencecompound. In some embodiments, alteration of core structure, for exampleby addition or removal of a small number of bonds (typically not morethan 5, 4, 3, 2, or 1 bonds, and often only a single bond) may generatea substituted compound equivalent to a parent reference compound. Inmany embodiments, equivalent compounds may be prepared by methodsillustrated in general reaction schemes as, for example, describedbelow, or by modifications thereof, using readily available startingmaterials, reagents and conventional or provided synthesis procedures.In these reactions, it is also possible to make use of variants, whichare in themselves known, but are not mentioned here.

Equivalent Dosage: The term “equivalent dosage” is used herein tocompare dosages of different pharmaceutically active agents that effectthe same biological result. Dosages of two different agents areconsidered to be “equivalent” to one another in accordance with thepresent invention if they achieve a comparable level or extent of thebiological result. In some embodiments, equivalent dosages of differentpharmaceutical agents for use in accordance with the present inventionare determined using in vitro and/or in vivo assays as described herein.In some embodiments, one or more lysosomal activating agents for use inaccordance with the present invention is utilized at a dose equivalentto a dose of a reference lysosomal activating agent; in some suchembodiments, the reference lysosomal activating agent for such purposeis selected from the group consisting of small molecule allostericactivators (e.g., pyrazolpyrimidines), imminosugars (e.g., isofagomine),antioxidants (e.g., n-acetyl-cysteine), and regulators of cellulartrafficking (e.g., Rab1a polypeptide).

Heteroaliphatic: The term “heteroaliphatic” refers to an aliphatic groupwherein one or more units selected from C, CH, CH₂, or CH₃ areindependently replaced by a heteroatom. In some embodiments, aheteroaliphatic group is heteroalkyl. In some embodiments, aheteroaliphatic group is heteroalkenyl.

Heteroaryl: The terms “heteroaryl” and “heteroar-,” used alone or aspart of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,”refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ringatoms; having 6, 10, or 14 π electrons shared in a cyclic array; andhaving, in addition to carbon atoms, from one to five heteroatoms. Theterm “heteroatom” refers to nitrogen, oxygen, or sulfur, and includesany oxidized form of nitrogen or sulfur, and any quaternized form of abasic nitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-,” as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring,” “heteroarylgroup,” or “heteroaromatic,” any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

Heteroatom: The term “heteroatom” means one or more of oxygen, sulfur,nitrogen, phosphorus, or silicon (including, any oxidized form ofnitrogen, sulfur, phosphorus, or silicon; the quaternized form of anybasic nitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

Heterocycle: As used herein, the terms “heterocycle,” “heterocyclyl,”“heterocyclic radical,” and “heterocyclic ring” are used interchangeablyand refer to a stable 3- to 7-membered monocyclic or 7-10-memberedbicyclic heterocyclic moiety that is either saturated or partiallyunsaturated, and having, in addition to carbon atoms, one or more,preferably one to four, heteroatoms, as defined above. When used inreference to a ring atom of a heterocycle, the term “nitrogen” includesa substituted nitrogen. As an example, in a saturated or partiallyunsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur ornitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (asin pyrrolidinyl), or NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclicgroup,” “heterocyclic moiety,” and “heterocyclic radical,” are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

Intraperitoneal: The phrases “intraperitoneal administration” and“administered intraperitonealy” as used herein have their art-understoodmeaning referring to administration of a compound or composition intothe peritoneum of a subject.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within an organism (e.g.,animal, plant, and/or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, and/or microbe).

Lower alkyl: The term “lower alkyl” refers to a C₁₋₄ straight orbranched alkyl group. Exemplary lower alkyl groups are methyl, ethyl,propyl, isopropyl, butyl, isobutyl, and tert-butyl.

Lower haloalkyl: The term “lower haloalkyl” refers to a C₁₋₄ straight orbranched alkyl group that is substituted with one or more halogen atoms.

Optionally substituted: As described herein, compounds of the inventionmay contain “optionally substituted” moieties. In general, the term“substituted,” whether preceded by the term “optionally” or not, meansthat one or more hydrogens of the designated moiety are replaced with asuitable substituent. Unless otherwise indicated, an “optionallysubstituted” group may have a suitable substituent at each substitutableposition of the group, and when more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. Combinations of substituents envisioned bythis invention are preferably those that result in the formation ofstable or chemically feasible compounds. The term “stable,” as usedherein, refers to compounds that are not substantially altered whensubjected to conditions to allow for their production, detection, and,in certain embodiments, their recovery, purification, and use for one ormore of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(∘); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘)₂; —(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘), —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘);—(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂;—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; —OP(O)(OR^(∘))₂; —SiR^(∘) ₃; —(C₁₋₄ straight or branchedalkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(o) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Oral: The phrases “oral administration” and “administered orally” asused herein have their art-understood meaning referring toadministration by mouth of a compound or composition.

Parenteral: The phrases “parenteral administration” and “administeredparenterally” as used herein have their art-understood meaning referringto modes of administration other than enteral and topicaladministration, usually by injection, and include, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid,intraspinal, and intrasternal injection and infusion.

Partially unsaturated: As used herein, the term “partially unsaturated”refers to a ring moiety that includes at least one double or triplebond. The term “partially unsaturated” is intended to encompass ringshaving multiple sites of unsaturation, but is not intended to includearyl or heteroaryl moieties, as herein defined.

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” refers to an active agent, formulated together with one ormore pharmaceutically acceptable carriers. In some embodiments, activeagent is present in unit dose amount appropriate for administration in atherapeutic regimen that shows a statistically significant probabilityof achieving a predetermined therapeutic effect when administered to arelevant population. In some embodiments, pharmaceutical compositionsmay be specially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream, or foam; sublingually; ocularly; transdermally; or nasally,pulmonary, and to other mucosal surfaces.

Pharmaceutically acceptable: As used herein, the phrase“pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Pharmaceutically acceptable carrier: As used herein, the term“pharmaceutically acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

Pharmaceutically acceptable salt: The term “pharmaceutically acceptablesalt”, as used herein, refers to salts of such compounds that areappropriate for use in pharmaceutical contexts, i.e., salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, S. M. Berge, et al. describespharmaceutically acceptable salts in detail in J. PharmaceuticalSciences, 66: 1-19 (1977). In some embodiments, pharmaceuticallyacceptable salt include, but are not limited to, nontoxic acid additionsalts, which are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. In someembodiments, pharmaceutically acceptable salts include, but are notlimited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. In someembodiments, pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms,sulfonate and aryl sulfonate.

Prodrug: A general, a “prodrug,” as that term is used herein and as isunderstood in the art, is an entity that, when administered to anorganism, is metabolized in the body to deliver an active (e.g.,therapeutic or diagnostic) agent of interest. Typically, such metabolisminvolves removal of at least one “prodrug moiety” so that the activeagent is formed. Various forms of “prodrugs” are known in the art. Forexamples of such prodrug moieties, see:

-   -   a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985)        and Methods in Enzymology, 42:309-396, edited by K. Widder, et        al. (Academic Press, 1985);    -   b) Prodrugs and Targeted Delivery, edited by by J. Rautio        (Wiley, 2011);    -   c) Prodrugs and Targeted Delivery, edited by by J. Rautio        (Wiley, 2011);    -   d) A Textbook of Drug Design and Development, edited by        Krogsgaard-Larsen;    -   e) Bundgaard, Chapter 5 “Design and Application of Prodrugs”,        by H. Bundgaard, p. 113-191 (1991);    -   f) Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992);    -   g) Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285        (1988); and    -   h) Kakeya, et al., Chem. Pharm. Bull., 32:692 (1984).

As with other compounds described herein, prodrugs may be provided inany of a variety of forms, e.g., crystal forms, salt forms etc. In someembodiments, prodrugs are provided as pharmaceutically acceptable saltsthereof.

Protecting group: The term “protecting group,” as used herein, is wellknown in the art and includes those described in detail in ProtectingGroups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd)edition, John Wiley & Sons, 1999, the entirety of which is incorporatedherein by reference. Also included are those protecting groups speciallyadapted for nucleoside and nucleotide chemistry described in CurrentProtocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al.June 2012, the entirety of Chapter 2 is incorporated herein byreference. Suitable amino-protecting groups include methyl carbamate,ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Suitably protected carboxylic acids further include, but are not limitedto, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylicacids. Examples of suitable silyl groups include trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,triisopropylsilyl, and the like. Examples of suitable alkyl groupsinclude methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl,t-butyl, tetrahydropyran-2-yl. Examples of suitable alkenyl groupsinclude allyl. Examples of suitable aryl groups include optionallysubstituted phenyl, biphenyl, or naphthyl. Examples of suitablearylalkyl groups include optionally substituted benzyl (e.g.,p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2-and 4-picolyl.

Suitable hydroxyl protecting groups include methyl, methoxylmethyl(MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkylN,N,N,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate,dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate,methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). Forprotecting 1,2- or 1,3-diols, the protecting groups include methyleneacetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylideneketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylideneacetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal,cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal,2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal,2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethyleneacetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester,1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho ester,α-methoxybenzylidene ortho ester, 1-(N,N-dimethylamino)ethylidenederivative, α-(N,N′-dimethylamino)benzylidene derivative,2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

In some embodiments, a hydroxyl protecting group is acetyl, t-butyl,t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl,diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl),4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl,trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate,tosylate, triflate, trityl, monomethoxytrityl (MMTr),4,4′-dimethoxytrityl, (DMTr) and 4,4′,4″-trimethoxytrityl (TMTr),2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE),2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl(NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl,2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl,2-(2-nitrophenyl)ethyl, butylthiocarbonyl,4,4′,4″-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl,2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl(Ptmt), 9-phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl(MOX). In some embodiments, each of the hydroxyl protecting groups is,independently selected from acetyl, benzyl, t-butyldimethylsilyl,t-butyldiphenylsilyl and 4,4′-dimethoxytrityl. In some embodiments, thehydroxyl protecting group is selected from the group consisting oftrityl, monomethoxytrityl and 4,4′-dimethoxytrityl group.

In some embodiments, a phosphorous protecting group is a group attachedto the internucleotide phosphorous linkage throughout oligonucleotidesynthesis. In some embodiments, the phosphorous protecting group isattached to the sulfur atom of the internucleotide phosphorothioatelinkage. In some embodiments, the phosphorous protecting group isattached to the oxygen atom of the internucleotide phosphorothioatelinkage. In some embodiments, the phosphorous protecting group isattached to the oxygen atom of the internucleotide phosphate linkage. Insome embodiments the phosphorous protecting group is 2-cyanoethyl (CE orCne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl,benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe),2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl,4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl,3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl,2-(N-formyl,N-methyl)aminoethyl,4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

Protein: As used herein, the term “protein” refers to a polypeptide(i.e., a string of at least two amino acids linked to one another bypeptide bonds). In some embodiments, proteins include onlynaturally-occurring amino acids. In some embodiments, proteins includeone or more non-naturally-occurring amino acids (e.g., moieties thatform one or more peptide bonds with adjacent amino acids). In someembodiments, one or more residues in a protein chain contain anon-amino-acid moiety (e.g., a glycan, etc). In some embodiments, aprotein includes more than one polypeptide chain, for example linked byone or more disulfide bonds or associated by other means. In someembodiments, proteins contain L-amino acids, D-amino acids, or both; insome embodiments, proteins contain one or more amino acid modificationsor analogs known in the art. Useful modifications include, e.g.,terminal acetylation, amidation, methylation, etc. The term “peptide” isgenerally used to refer to a polypeptide having a length of less thanabout 100 amino acids, less than about 50 amino acids, less than 20amino acids, or less than 10 amino acids. In some embodiments, proteinsare antibodies, antibody fragments, biologically active portionsthereof, and/or characteristic portions thereof.

Sample: As used herein, the term “sample” refers to a biological sampleobtained or derived from a source of interest, as described herein. Insome embodiments, a source of interest comprises an organism, such as ananimal or human. In some embodiments, a biological sample comprisesbiological tissue or fluid. In some embodiments, a biological sample isor comprises bone marrow; blood; blood cells; ascites; tissue or fineneedle biopsy samples; cell-containing body fluids; free floatingnucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritonealfluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs;vaginal swabs; oral swabs; nasal swabs; washings or lavages such as aductal lavages or bronchioalveolar lavages; aspirates; scrapings; bonemarrow specimens; tissue biopsy specimens; surgical specimens; feces,other body fluids, secretions, and/or excretions; and/or cellstherefrom, etc. In some embodiments, a biological sample is or comprisescells obtained from an individual. In some embodiments, a sample is a“primary sample” obtained directly from a source of interest by anyappropriate means. For example, in some embodiments, a primarybiological sample is obtained by methods selected from the groupconsisting of biopsy (e.g., fine needle aspiration or tissue biopsy),surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc.In some embodiments, as will be clear from context, the term “sample”refers to a preparation that is obtained by processing (e.g., byremoving one or more components of and/or by adding one or more agentsto) a primary sample. For example, filtering using a semi-permeablemembrane. Such a “processed sample” may comprise, for example nucleicacids or proteins extracted from a sample or obtained by subjecting aprimary sample to techniques such as amplification or reversetranscription of mRNA, isolation and/or purification of certaincomponents, etc.

Stereochemically isomeric forms: The phrase “stereochemically isomericforms,” as used herein, refers to different compounds made up of thesame atoms bonded by the same sequence of bonds but having differentthree-dimensional structures which are not interchangeable. In someembodiments of the invention, provided chemical compositions may be orinclude pure preparations of individual stereochemically isomeric formsof a compound; in some embodiments, provided chemical compositions maybe or include mixtures of two or more stereochemically isomeric forms ofthe compound. In certain embodiments, such mixtures contain equalamounts of different stereochemically isomeric forms; in certainembodiments, such mixtures contain different amounts of at least twodifferent stereochemically isomeric forms. In some embodiments, achemical composition may contain all diastereomers and/or enantiomers ofthe compound. In some embodiments, a chemical composition may containless than all diastereomers and/or enantiomers of a compound. In someembodiments, if a particular enantiomer of a compound of the presentinvention is desired, it may be prepared, for example, by asymmetricsynthesis, or by derivation with a chiral auxiliary, where the resultingdiastereomeric mixture is separated and the auxiliary group cleaved toprovide the pure desired enantiomers. Alternatively, where the moleculecontains a basic functional group, such as amino, diastereomeric saltsare formed with an appropriate optically-active acid, and resolved, forexample, by fractional crystallization.

Subject: As used herein, the term “subject” or “test subject” refers toany organism to which a provided compound or composition is administeredin accordance with the present invention e.g., for experimental,diagnostic, prophylactic, and/or therapeutic purposes. Typical subjectsinclude animals (e.g., mammals such as mice, rats, rabbits, non-humanprimates, and humans; insects; worms; etc.) and plants. In someembodiments, a subject may be suffering from, and/or susceptible to adisease, disorder, and/or condition.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and/or chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with and/or displays oneor more symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition is one who has a higher risk of developingthe disease, disorder, and/or condition than does a member of thegeneral public. In some embodiments, an individual who is susceptible toa disease, disorder and/or condition may not have been diagnosed withthe disease, disorder, and/or condition. In some embodiments, anindividual who is susceptible to a disease, disorder, and/or conditionmay exhibit symptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition may not exhibit symptoms of the disease, disorder,and/or condition. In some embodiments, an individual who is susceptibleto a disease, disorder, and/or condition will develop the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will not developthe disease, disorder, and/or condition.

Systemic: The phrases “systemic administration,” “administeredsystemically,” “peripheral administration,” and “administeredperipherally” as used herein have their art-understood meaning referringto administration of a compound or composition such that it enters therecipient's system.

Tautomeric forms: The phrase “tautomeric forms,” as used herein, is usedto describe different isomeric forms of organic compounds that arecapable of facile interconversion. Tautomers may be characterized by theformal migration of a hydrogen atom or proton, accompanied by a switchof a single bond and adjacent double bond. In some embodiments,tautomers may result from prototropic tautomerism (i.e., the relocationof a proton). In some embodiments, tautomers may result from valencetautomerism (i.e., the rapid reorganization of bonding electrons). Allsuch tautomeric forms are intended to be included within the scope ofthe present invention. In some embodiments, tautomeric forms of acompound exist in mobile equilibrium with each other, so that attemptsto prepare the separate substances results in the formation of amixture. In some embodiments, tautomeric forms of a compound areseparable and isolatable compounds. In some embodiments of theinvention, chemical compositions may be provided that are or includepure preparations of a single tautomeric form of a compound. In someembodiments of the invention, chemical compositions may be provided asmixtures of two or more tautomeric forms of a compound. In certainembodiments, such mixtures contain equal amounts of different tautomericforms; in certain embodiments, such mixtures contain different amountsof at least two different tautomeric forms of a compound. In someembodiments of the invention, chemical compositions may contain alltautomeric forms of a compound. In some embodiments of the invention,chemical compositions may contain less than all tautomeric forms of acompound. In some embodiments of the invention, chemical compositionsmay contain one or more tautomeric forms of a compound in amounts thatvary over time as a result of interconversion. In some embodiments ofthe invention, the tautomerism is keto-enol tautomerism. One of skill inthe chemical arts would recognize that a keto-enol tautomer can be“trapped” (i.e., chemically modified such that it remains in the “enol”form) using any suitable reagent known in the chemical arts in toprovide an enol derivative that may subsequently be isolated using oneor more suitable techniques known in the art. Unless otherwiseindicated, the present invention encompasses all tautomeric forms ofrelevant compounds, whether in pure form or in admixture with oneanother.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that, when administered to a subject, has a therapeuticeffect and/or elicits a desired biological and/or pharmacologicaleffect. In some embodiments, a therapeutic agent is any substance thatcan be used to alleviate, ameliorate, relieve, inhibit, prevent, delayonset of, reduce severity of, and/or reduce incidence of one or moresymptoms or features of a disease, disorder, and/or condition.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of a substance (e.g.,a therapeutic agent, composition, and/or formulation) that elicits adesired biological response when administered as part of a therapeuticregimen. In some embodiments, a therapeutically effective amount of asubstance is an amount that is sufficient, when administered to asubject suffering from or susceptible to a disease, disorder, and/orcondition, to treat, diagnose, prevent, and/or delay the onset of thedisease, disorder, and/or condition. As will be appreciated by those ofordinary skill in this art, the effective amount of a substance may varydepending on such factors as the desired biological endpoint, thesubstance to be delivered, the target cell or tissue, etc. For example,the effective amount of compound in a formulation to treat a disease,disorder, and/or condition is the amount that alleviates, ameliorates,relieves, inhibits, prevents, delays onset of, reduces severity ofand/or reduces incidence of one or more symptoms or features of thedisease, disorder, and/or condition. In some embodiments, atherapeutically effective amount is administered in a single dose; insome embodiments, multiple unit doses are required to deliver atherapeutically effective amount.

Treat: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof, and/or reduce incidence of one or more symptoms or features of adisease, disorder, and/or condition. Treatment may be administered to asubject who does not exhibit signs of a disease, disorder, and/orcondition. In some embodiments, treatment may be administered to asubject who exhibits only early signs of the disease, disorder, and/orcondition, for example for the purpose of decreasing the risk ofdeveloping pathology associated with the disease, disorder, and/orcondition.

Unsaturated: The term “unsaturated,” as used herein, means that a moietyhas one or more units of unsaturation.

Unit dose: The expression “unit dose” as used herein refers to an amountadministered as a single dose and/or in a physically discrete unit of apharmaceutical composition. In many embodiments, a unit dose contains apredetermined quantity of an active agent. In some embodiments, a unitdose contains an entire single dose of the agent. In some embodiments,more than one unit dose is administered to achieve a total single dose.In some embodiments, administration of multiple unit doses is required,or expected to be required, in order to achieve an intended effect. Aunit dose may be, for example, a volume of liquid (e.g., an acceptablecarrier) containing a predetermined quantity of one or more therapeuticagents, a predetermined amount of one or more therapeutic agents insolid form, a sustained release formulation or drug delivery devicecontaining a predetermined amount of one or more therapeutic agents,etc. It will be appreciated that a unit dose may be present in aformulation that includes any of a variety of components in addition tothe therapeutic agent(s). For example, acceptable carriers (e.g.,pharmaceutically acceptable carriers), diluents, stabilizers, buffers,preservatives, etc., may be included as described infra. It will beappreciated by those skilled in the art, in many embodiments, a totalappropriate daily dosage of a particular therapeutic agent may comprisea portion, or a plurality, of unit doses, and may be decided, forexample, by the attending physician within the scope of sound medicaljudgment. In some embodiments, the specific effective dose level for anyparticular subject or organism may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of specific active compound employed; specific compositionemployed; age, body weight, general health, sex and diet of the subject;time of administration, and rate of excretion of the specific activecompound employed; duration of the treatment; drugs and/or additionaltherapies used in combination or coincidental with specific compound(s)employed, and like factors well known in the medical arts.

Wild-type: As used herein, the term “wild-type” has its art-understoodmeaning that refers to an entity having a structure and/or activity asfound in nature in a “normal” (as contrasted with mutant, diseased,altered, etc) state or context. Those of ordinary skill in the art willappreciate that wild type genes and polypeptides often exist in multipledifferent forms (e.g., alleles).

Nucleic acid: The term “nucleic acid” includes any nucleotides, analogsthereof, and polymers thereof. The term “polynucleotide” as used hereinrefer to a polymeric form of nucleotides of any length, eitherribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms referto the primary structure of the molecules and, thus, include double- andsingle-stranded DNA, and double- and single-stranded RNA. These termsinclude, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs and modified polynucleotides such as, though notlimited to, methylated, protected and/or capped nucleotides orpolynucleotides. The terms encompass poly- or oligo-ribonucleotides(RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derivedfrom N-glycosides or C-glycosides of nucleobases and/or modifiednucleobases; nucleic acids derived from sugars and/or modified sugars;and nucleic acids derived from phosphate bridges and/or modifiedphosphorus-atom bridges (also referred to herein as “internucleotidelinkages”). The term encompasses nucleic acids containing anycombinations of nucleobases, modified nucleobases, sugars, modifiedsugars, phosphate bridges or modified phosphorus atom bridges. Examplesinclude, and are not limited to, nucleic acids containing ribosemoieties, the nucleic acids containing deoxy-ribose moieties, nucleicacids containing both ribose and deoxyribose moieties, nucleic acidscontaining ribose and modified ribose moieties. The prefix poly- refersto a nucleic acid containing 2 to about 10,000 nucleotide monomer unitsand wherein the prefix oligo- refers to a nucleic acid containing 2 toabout 200 nucleotide monomer units.

Nucleotide: The term “nucleotide” as used herein refers to a monomericunit of a polynucleotide that consists of a heterocyclic base, a sugar,and one or more phosphate groups or phosphorus-containinginternucleotidic linkages. The naturally occurring bases, (guanine, (G),adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) arederivatives of purine or pyrimidine, though it should be understood thatnaturally and non-naturally occurring base analogs are also included.The naturally occurring sugar is the pentose (five-carbon sugar)deoxyribose (which forms DNA) or ribose (which forms RNA), though itshould be understood that naturally and non-naturally occurring sugaranalogs are also included. Nucleotides are linked via internucleotidiclinkages to form nucleic acids, or polynucleotides. Manyinternucleotidic linkages are known in the art (such as, though notlimited to, phosphate, phosphorothioates, boranophosphates and thelike). Artificial nucleic acids include PNAs (peptide nucleic acids),phosphotriesters, phosphorothionates, H-phosphonates, phosphoramidates,boranophosphates, methylphosphonates, phosphonoacetates,thiophosphonoacetates and other variants of the phosphate backbone ofnative nucleic acids, such as those described herein.

Nucleoside: The term “nucleoside” refers to a moiety wherein anucleobase or a modified nucleobase is covalently bound to a sugar ormodified sugar.

Sugar: The term “sugar” refers to a monosaccharide in closed and/or openform. Sugars include, but are not limited to, ribose, deoxyribose,pentofuranose, pentopyranose, and hexopyranose moieties. As used herein,the term also encompasses structural analogs used in lieu ofconventional sugar molecules, such as glycol, polymer of which forms thebackbone of the nucleic acid analog, glycol nucleic acid (“GNA”).

Modified sugar: The term “modified sugar” refers to a moiety that canreplace a sugar. The modified sugar mimics the spatial arrangement,electronic properties, or some other physicochemical property of asugar.

Nucleobase: The term “nucleobase” refers to the parts of nucleic acidsthat are involved in the hydrogen-bonding that binds one nucleic acidstrand to another complementary strand in a sequence specific manner.The most common naturally-occurring nucleobases are adenine (A), guanine(G), uracil (U), cytosine (C), and thymine (T). In some embodiments, thenaturally-occurring nucleobases are modified adenine, guanine, uracil,cytosine, or thymine. In some embodiments, the naturally-occurringnucleobases are methylated adenine, guanine, uracil, cytosine, orthymine. In some embodiments, a nucleobase is a “modified nucleobase,”e.g., a nucleobase other than adenine (A), guanine (G), uracil (U),cytosine (C), and thymine (T). In some embodiments, the modifiednucleobases are methylated adenine, guanine, uracil, cytosine, orthymine. In some embodiments, the modified nucleobase mimics the spatialarrangement, electronic properties, or some other physicochemicalproperty of the nucleobase and retains the property of hydrogen-bondingthat binds one nucleic acid strand to another in a sequence specificmanner. In some embodiments, a modified nucleobase can pair with all ofthe five naturally occurring bases (uracil, thymine, adenine, cytosine,or guanine) without substantially affecting the melting behavior,recognition by intracellular enzymes or activity of the oligonucleotideduplex.

Chiral ligand: The term “chiral ligand” or “chiral auxiliary” refers toa moiety that is chiral and can be incorporated into a reaction so thatthe reaction can be carried out with certain stereoselectivity.

Condensing reagent: In a condensation reaction, the term “condensingreagent” refers to a reagent that activates a less reactive site andrenders it more susceptible to attack by another reagent. In someembodiments, such another reagent is a nucleophile.

Blocking group: The term “blocking group” refers to a group that masksthe reactivity of a functional group. The functional group can besubsequently unmasked by removal of the blocking group. In someembodiments, a blocking group is a protecting group.

Moiety: The term “moiety” refers to a specific segment or functionalgroup of a molecule. Chemical moieties are often recognized chemicalentities embedded in or appended to a molecule.

Solid support: The term “solid support” refers to any support whichenables synthesis of nucleic acids. In some embodiments, the term refersto a glass or a polymer, that is insoluble in the media employed in thereaction steps performed to synthesize nucleic acids, and is derivatizedto comprise reactive groups. In some embodiments, the solid support isHighly Cross-linked Polystyrene (HCP) or Controlled Pore Glass (CPG). Insome embodiments, the solid support is Controlled Pore Glass (CPG). Insome embodiments, the solid support is hybrid support of Controlled PoreGlass (CPG) and Highly Cross-linked Polystyrene (HCP).

Linking moiety: The term “linking moiety” refers to any moietyoptionally positioned between the terminal nucleoside and the solidsupport or between the terminal nucleoside and another nucleoside,nucleotide, or nucleic acid.

DNA molecule: A “DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itseither single stranded form or a double-stranded helix. This term refersonly to the primary and secondary structure of the molecule, and doesnot limit it to any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences can be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA).

Coding sequence: A DNA “coding sequence” or “coding region” is adouble-stranded DNA sequence which is transcribed and translated into apolypeptide in vivo when placed under the control of appropriateexpression control sequences. The boundaries of the coding sequence (the“open reading frame” or “ORF”) are determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, but is not limited to,prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequencesfrom eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence is,usually, be located 3′ to the coding sequence. The term “non-codingsequence” or “non-coding region” refers to regions of a polynucleotidesequence that are not translated into amino acids (e.g. 5′ and 3′un-translated regions).

Reading frame: The term “reading frame” refers to one of the sixpossible reading frames, three in each direction, of the double strandedDNA molecule. The reading frame that is used determines which codons areused to encode amino acids within the coding sequence of a DNA molecule.

Antisense: As used herein, an “antisense” nucleic acid moleculecomprises a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule, complementary to an mRNAsequence or complementary to the coding strand of a gene. Accordingly,an antisense nucleic acid molecule can associate via hydrogen bonds to asense nucleic acid molecule.

Wobble position: As used herein, a “wobble position” refers to the thirdposition of a codon. Mutations in a DNA molecule within the wobbleposition of a codon, in some embodiments, result in silent orconservative mutations at the amino acid level. For example, there arefour codons that encode Glycine, i.e., GGU, GGC, GGA and GGG, thusmutation of any wobble position nucleotide, to any other nucleotideselected from A, U, C and G, does not result in a change at the aminoacid level of the encoded protein and, therefore, is a silentsubstitution.

Silent substitution: a “silent substitution” or “silent mutation” is onein which a nucleotide within a codon is modified, but does not result ina change in the amino acid residue encoded by the codon. Examplesinclude mutations in the third position of a codon, as well in the firstposition of certain codons such as in the codon “CGG” which, whenmutated to AGG, still encodes Arg.

Gene: The terms “gene,” “recombinant gene” and “gene construct” as usedherein, refer to a DNA molecule, or portion of a DNA molecule, thatencodes a protein or a portion thereof. The DNA molecule can contain anopen reading frame encoding the protein (as exon sequences) and canfurther include intron sequences. The term “intron” as used herein,refers to a DNA sequence present in a given gene which is not translatedinto protein and is found in some, but not all cases, between exons. Itcan be desirable for the gene to be operably linked to, (or it cancomprise), one or more promoters, enhancers, repressors and/or otherregulatory sequences to modulate the activity or expression of the gene,as is well known in the art.

Complementary DNA: As used herein, a “complementary DNA” or “cDNA”includes recombinant polynucleotides synthesized by reversetranscription of mRNA and from which intervening sequences (introns)have been removed.

Homology: “Homology” or “identity” or “similarity” refers to sequencesimilarity between two nucleic acid molecules. Homology and identity caneach be determined by comparing a position in each sequence which can bealigned for purposes of comparison. When an equivalent position in thecompared sequences is occupied by the same base, then the molecules areidentical at that position; when the equivalent site occupied by thesame or a similar nucleic acid residue (e.g., similar in steric and/orelectronic nature), then the molecules can be referred to as homologous(similar) at that position. Expression as a percentage ofhomology/similarity or identity refers to a function of the number ofidentical or similar nucleic acids at positions shared by the comparedsequences. A sequence which is “unrelated” or “non-homologous” sharesless than 40% identity, less than 35% identity, less than 30% identity,or less than 25% identity with a sequence described herein. In comparingtwo sequences, the absence of residues (amino acids or nucleic acids) orpresence of extra residues also decreases the identity andhomology/similarity.

In some embodiments, the term “homology” describes a mathematicallybased comparison of sequence similarities which is used to identifygenes with similar functions or motifs. The nucleic acid sequencesdescribed herein can be used as a “query sequence” to perform a searchagainst public databases, for example, to identify other family members,related sequences or homologs. In some embodiments, such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. In some embodiments,BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12 to obtain nucleotide sequences homologous tonucleic acid molecules of the invention. In some embodiments, to obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and BLAST)can be used (See www.ncbi.nlm.nih.gov).

Identity: As used herein, “identity” means the percentage of identicalnucleotide residues at corresponding positions in two or more sequenceswhen the sequences are aligned to maximize sequence matching, i.e.,taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990). The well-known Smith Watermanalgorithm can also be used to determine identity.

Heterologous: A “heterologous” region of a DNA sequence is anidentifiable segment of DNA within a larger DNA sequence that is notfound in association with the larger sequence in nature. Thus, when theheterologous region encodes a mammalian gene, the gene can usually beflanked by DNA that does not flank the mammalian genomic DNA in thegenome of the source organism. Another example of a heterologous codingsequence is a sequence where the coding sequence itself is not found innature (e.g., a cDNA where the genomic coding sequence contains intronsor synthetic sequences having codons or motifs different than theunmodified gene). Allelic variations or naturally-occurring mutationalevents do not give rise to a heterologous region of DNA as definedherein.

Transition mutation: The term “transition mutations” refers to basechanges in a DNA sequence in which a pyrimidine (cytidine (C) orthymidine (T) is replaced by another pyrimidine, or a purine (adenosine(A) or guanosine (G) is replaced by another purine.

Transversion mutation: The term “transversion mutations” refers to basechanges in a DNA sequence in which a pyrimidine (cytidine (C) orthymidine (T) is replaced by a purine (adenosine (A) or guanosine (G),or a purine is replaced by a pyrimidine.

Oligonucleotide: the term “oligonucleotide” refers to a polymer oroligomer of nucleotide monomers, containing any combination ofnucleobases, modified nucleobases, sugars, modified sugars, phosphatebridges, or modified phosphorus atom bridges (also referred to herein as“internucleotidic linkage”, defined further herein).

Oligonucleotides can be single-stranded or double-stranded. As usedherein, the term “oligonucleotide strand” encompasses a single-strandedoligonucleotide. A single-stranded oligonucleotide can havedouble-stranded regions and a double-stranded oligonucleotide can havesingle-stranded regions. Exemplary oligonucleotides include, but are notlimited to structural genes, genes including control and terminationregions, self-replicating systems such as viral or plasmid DNA,single-stranded and double-stranded siRNAs and other RNA interferencereagents (RNAi agents or iRNA agents), shRNA, antisenseoligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs,aptamers, antimirs, antagomirs, U adaptors, triplex-formingoligonucleotides, G-quadruplex oligonucleotides, RNA activators,immuno-stimulatory oligonucleotides, and decoy oligonucleotides.

Double-stranded and single-stranded oligonucleotides that are effectivein inducing RNA interference are also referred to as siRNA, RNAi agent,or iRNA agent, herein. In some embodiments, these RNA interferenceinducing oligonucleotides associate with a cytoplasmic multi-proteincomplex known as RNAi-induced silencing complex (RISC). In manyembodiments, single-stranded and double-stranded RNAi agents aresufficiently long that they can be cleaved by an endogenous molecule,e.g., by Dicer, to produce smaller oligonucleotides that can enter theRISC machinery and participate in RISC mediated cleavage of a targetsequence, e.g. a target mRNA.

Oligonucleotides of the present invention can be of various lengths. Inparticular embodiments, oligonucleotides can range from about 2 to about200 nucleotides in length. In various related embodiments,oligonucleotides, single-stranded, double-stranded, and triple-stranded,can range in length from about 4 to about 10 nucleotides, from about 10to about 50 nucleotides, from about 20 to about 50 nucleotides, fromabout 15 to about 30 nucleotides, from about 20 to about 30 nucleotidesin length. In some embodiments, the oligonucleotide is from about 9 toabout 39 nucleotides in length. In some embodiments, the oligonucleotideis at least 4 nucleotides in length. In some embodiments, theoligonucleotide is at least 5 nucleotides in length. In someembodiments, the oligonucleotide is at least 6 nucleotides in length. Insome embodiments, the oligonucleotide is at least 7 nucleotides inlength. In some embodiments, the oligonucleotide is at least 8nucleotides in length. In some embodiments, the oligonucleotide is atleast 9 nucleotides in length. In some embodiments, the oligonucleotideis at least 10 nucleotides in length. In some embodiments, theoligonucleotide is at least 11 nucleotides in length. In someembodiments, the oligonucleotide is at least 12 nucleotides in length.In some embodiments, the oligonucleotide is at least 15 nucleotides inlength. In some embodiments, the oligonucleotide is at least 20nucleotides in length. In some embodiments, the oligonucleotide is atleast 25 nucleotides in length. In some embodiments, the oligonucleotideis at least 30 nucleotides in length. In some embodiments, theoligonucleotide is a duplex of complementary strands of at least 18nucleotides in length. In some embodiments, the oligonucleotide is aduplex of complementary strands of at least 21 nucleotides in length.

Internucleotidic linkage: As used herein, the phrase “internucleotidiclinkage” refers generally to the phosphorus-containing linkage betweennucleotide units of an oligonucleotide, and is interchangeable with“inter-sugar linkage” and “phosphorus atom bridge,” as used above andherein. In some embodiments, an internucleotidic linkage is aphosphodiester linkage, as found in naturally occurring DNA and RNAmolecules. In some embodiments, an internucleotidic linkage is a“modified internucleotidic linkage” wherein each oxygen atom of thephosphodiester linkage is optionally and independently replaced by anorganic or inorganic moiety. In some embodiments, such an organic orinorganic moiety is selected from but not limited to ═S, ═Se, ═NR′,—SR′, —SeR′, —N(R′)₂, B(R′)₃, —S—, —Se—, and —N(R′)—, wherein each R′ isindependently as defined and described below. In some embodiments, aninternucleotidic linkage is a phosphotriester linkage, phosphorothioatediester linkage

or modified phosphorothioate triester linkage. It is understood by aperson of ordinary skill in the art that the internucleotidic linkagemay exist as an anion or cation at a given pH due to the existence ofacid or base moieties in the linkage.

Unless otherwise specified, when used with an oligonucleotide sequence,each of s, s1, s2, s3, s4, s5, s6 and s7 independently represents thefollowing modified internucleotidic linkage as illustrated in Table 1,below.

TABLE 1 Exemplary Modified Internucleotidic Linkage. Sym- bol ModifiedInternucleotidic Linkage s

s1

s2

s3

s4

s5

s6

s7

s8

s9

s10

s11

s12

s13

s14

s15

s16

s17

s18

For instance, (Rp, Sp)-ATsCs1GA has 1) a phosphorothioateinternucleotidic linkage

between T and C; and 2) a phosphorothioate triester internucleotidiclinkage having the structure of

between C and G. Unless otherwise specified, the Rp/Sp designationspreceding an oligonucleotide sequence describe the configurations ofchiral linkage phosphorus atoms in the internucleotidic linkagessequentially from 5′ to 3′ of the oligonucleotide sequence. Forinstance, in (Rp, Sp)-ATsCs1GA, the phosphorus in the “s” linkagebetween T and C has Rp configuration and the phosphorus in “s1” linkagebetween C and G has Sp configuration. In some embodiments, “All-(Rp)” or“All-(Sp)” is used to indicate that all chiral linkage phosphorus atomsin oligonucleotide have the same Rp or Sp configuration, respectively.For instance, All-(Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ IDNO: 106) indicates that all the chiral linkage phosphorus atoms in theoligonucleotide have Rp configuration;All-(Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO: 106)indicates that all the chiral linkage phosphorus atoms in theoligonucleotide have Sp configuration.

Oligonucleotide type: As used herein, the phrase “oligonucleotide type”is used to define an oligonucleotide that has a particular basesequence, pattern of backbone linkages (i.e., pattern ofinternucleotidic linkage types, for example, phosphate,phosphorothioate, etc), pattern of backbone chiral centers (i.e. patternof linkage phosphorus stereochemistry (Rp/Sp)), and pattern of backbonephosphorus modifications (e.g., pattern of “—XLR¹” groups in formula I).Oligonucleotides of a common designated “type” are structurallyidentical to one another.

One of skill in the art will appreciate that synthetic methods of thepresent invention provide for a degree of control during the synthesisof an oligonucleotide strand such that each nucleotide unit of theoligonucleotide strand can be designed and/or selected in advance tohave a particular stereochemistry at the linkage phosphorus and/or aparticular modification at the linkage phosphorus, and/or a particularbase, and/or a particular sugar. In some embodiments, an oligonucleotidestrand is designed and/or selected in advance to have a particularcombination of stereocenters at the linkage phosphorus. In someembodiments, an oligonucleotide strand is designed and/or determined tohave a particular combination of modifications at the linkagephosphorus. In some embodiments, an oligonucleotide strand is designedand/or selected to have a particular combination of bases. In someembodiments, an oligonucleotide strand is designed and/or selected tohave a particular combination of one or more of the above structuralcharacteristics. The present invention provides compositions comprisingor consisting of a plurality of oligonucleotide molecules (e.g.,chirally controlled oligonucleotide compositions). In some embodiments,all such molecules are of the same type (i.e., are structurallyidentical to one another). In many embodiments, however, providedcompositions comprise a plurality of oligonucleotides of differenttypes, typically in pre-determined relative amounts.

Chiral control: As used herein, “chiral control” refers to an ability tocontrol the stereochemical designation of every chiral linkagephosphorus within an oligonucleotide strand. The phrase “chirallycontrolled oligonucleotide” refers to an oligonucleotide which exists ina single diastereomeric form with respect to the chiral linkagephosphorus.

Chirally controlled oligonucleotide composition: As used herein, thephrase “chirally controlled oligonucleotide composition” refers to anoligonucleotide composition that contains predetermined levels ofindividual oligonucleotide types. For instance, in some embodiments achirally controlled oligonucleotide composition comprises oneoligonucleotide type. In some embodiments, a chirally controlledoligonucleotide composition comprises more than one oligonucleotidetype. In some embodiments, a chirally controlled oligonucleotidecomposition comprises a mixture of multiple oligonucleotide types.Exemplary chirally controlled oligonucleotide compositions are describedfurther herein.

Chirally pure: as used herein, the phrase “chirally pure” is used todescribe a chirally controlled oligonucleotide composition in which allof the oligonucleotides exist in a single diastereomeric form withrespect to the linkage phosphorus.

Chirally uniform: as used herein, the phrase “chirally uniform” is usedto describe an oligonucleotide molecule or type in which all nucleotideunits have the same stereochemistry at the linkage phosphorus. Forinstance, an oligonucleotide whose nucleotide units all have Rpstereochemistry at the linkage phosphorus is chirally uniform. Likewise,an oligonucleotide whose nucleotide units all have Sp stereochemistry atthe linkage phosphorus is chirally uniform.

Predetermined: By predetermined is meant deliberately selected, forexample as opposed to randomly occurring or achieved. Those of ordinaryskill in the art, reading the present specification, will appreciatethat the present invention provides new and surprising technologies thatpermit selection of particular oligonucleotide types for preparationand/or inclusion in provided compositions, and further permitscontrolled preparation of precisely the selected particular types,optionally in selected particular relative amounts, so that providedcompositions are prepared. Such provided compositions are“predetermined” as described herein. Compositions that may containcertain individual oligonucleotide types because they happen to havebeen generated through a process that cannot be controlled tointentionally generate the particular oligonucleotide types is not a“predetermined” composition. In some embodiments, a predeterminedcomposition is one that can be intentionally reproduced (e.g., throughrepetition of a controlled process).

Linkage phosphorus: as defined herein, the phrase “linkage phosphorus”is used to indicate that the particular phosphorus atom being referredto is the phosphorus atom present in the internucleotidic linkage, whichphosphorus atom corresponds to the phosphorus atom of a phosphodiesterof an internucleotidic linkage as occurs in naturally occurring DNA andRNA. In some embodiments, a linkage phosphorus atom is in a modifiedinternucleotidic linkage, wherein each oxygen atom of a phosphodiesterlinkage is optionally and independently replaced by an organic orinorganic moiety. In some embodiments, a linkage phosphorus atom is P*of formula I. In some embodiments, a linkage phosphorus atom is chiral.In some embodiments, a chiral linkage phosphorus atom is P* of formulaI.

P-modification: as used herein, the term “P-modification” refers to anymodification at the linkage phosphorus other than a stereochemicalmodification. In some embodiments, a P-modification comprises addition,substitution, or removal of a pendant moiety covalently attached to alinkage phosphorus. In some embodiments, the “P-modification” is —X-L-R¹wherein each of X, L and R¹ is independently as defined and describedherein and below.

Blockmer: the term “blockmer,” as used herein, refers to anoligonucleotide strand whose pattern of structural featurescharacterizing each individual nucleotide unit is characterized by thepresence of at least two consecutive nucleotide units sharing a commonstructural feature at the internucleotidic phosphorus linkage. By commonstructural feature is meant common stereochemistry at the linkagephosphorus or a common modification at the linkage phosphorus. In someembodiments, the at least two consecutive nucleotide units sharing acommon structure feature at the internucleotidic phosphorus linkage arereferred to as a “block”.

In some embodiments, a blockmer is a “stereoblockmer,” e.g., at leasttwo consecutive nucleotide units have the same stereochemistry at thelinkage phosphorus. Such at lest two consecutive nucleotide units form a“stereoblock.” For instance, (Sp, Sp)-ATsCs1GA is a stereoblockmerbecause at least two consecutive nucleotide units, the Ts and the Cs1,have the same stereochemistry at the linkage phosphorus (both Sp). Inthe same oligonucleotide (Sp, Sp)-ATsCs1GA, TsCs1 forms a block, and itis a stereoblock.

In some embodiments, a blockmer is a “P-modification blockmer,” e.g., atleast two consecutive nucleotide units have the same modification at thelinkage phosphorus. Such at lest two consecutive nucleotide units form a“P-modification block”. For instance, (Rp, Sp)-ATsCsGA is aP-modification blockmer because at least two consecutive nucleotideunits, the Ts and the Cs, have the same P-modification (i.e., both are aphosphorothioate diester). In the same oligonucleotide of (Rp,Sp)-ATsCsGA, TsCs forms a block, and it is a P-modification block.

In some embodiments, a blockmer is a “linkage blockmer,” e.g., at leasttwo consecutive nucleotide units have identical stereochemistry andidentical modifications at the linkage phosphorus. At least twoconsecutive nucleotide units form a “linkage block”. For instance, (Rp,Rp)-ATsCsGA is a linkage blockmer because at least two consecutivenucleotide units, the Ts and the Cs, have the same stereochemistry (bothRp) and P-modification (both phosphorothioate). In the sameoligonucleotide of (Rp, Rp)-ATsCsGA, TsCs forms a block, and it is alinkage block.

In some embodiments, a blockmer comprises one or more blocksindependently selected from a stereoblock, a P-modification block and alinkage block. In some embodiments, a blockmer is a stereoblockmer withrespect to one block, and/or a P-modification blockmer with respect toanother block, and/or a linkage blockmer with respect to yet anotherblock. For instance, (Rp, Rp, Rp, Rp, Rp, Sp, Sp,Sp)-AAsTsCsGsAs1Ts1Cs1Gs1ATCG (SEQ ID NO: 107) is a stereoblockmer withrespect to the stereoblock AsTsCsGsAs1 (all Rp at linkage phosphorus) orTs1Cs1Gs1 (all Sp at linkage phosphorus), a P-modification blockmer withrespect to the P-modification block AsTsCsGs (all s linkage) orAs1Ts1Cs1Gs1 (all s1 linkage), or a linkage blockmer with respect to thelinkage block AsTsCsGs (all Rp at linkage phosphorus and all s linkage)or Ts1Cs1Gs1 (all Sp at linkage phosphorus and all s1 linkage).

Altmer: the term “altmer,” as used herein, refers to an oligonucleotidestrand whose pattern of structural features characterizing eachindividual nucleotide unit is characterized in that no two consecutivenucleotide units of the oligonucleotide strand share a particularstructural feature at the internucleotidic phosphorus linkage. In someembodiments, an altmer is designed such that it comprises a repeatingpattern. In some embodiments, an altmer is designed such that it doesnot comprise a repeating pattern.

In some embodiments, an altmer is a “stereoaltmer,” e.g., no twoconsecutive nucleotide units have the same stereochemistry at thelinkage phosphorus. For instance, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO: 106).

In some embodiments, an altmer is a “P-modification altmer” e.g., no twoconsecutive nucleotide units have the same modification at the linkagephosphorus. For instance, All-(Sp)-CAs1GsT, in which each linkagephosphorus has a different P-modification than the others.

In some embodiments, an altmer is a “linkage altmer,” e.g., no twoconsecutive nucleotide units have identical stereochemistry or identicalmodifications at the linkage phosphorus. For instance, (Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp)-GsCs1CsTs1CsAs1GsTs1CsTs1GsCs1TsTs2CsGs3CsAs4CsC (SEQ ID NO: 106).

Unimer: the term “unimer,” as used herein, refers to an oligonucleotidestrand whose pattern of structural features characterizing eachindividual nucleotide unit is such that all nucleotide units within thestrand share at least one common structural feature at theinternucleotidic phosphorus linkage. By common structural feature ismeant common stereochemistry at the linkage phosphorus or a commonmodification at the linkage phosphorus.

In some embodiments, a unimer is a “stereounimer,” e.g., all nucleotideunits have the same stereochemistry at the linkage phosphorus. Forinstance, All-(Sp)-CsAs1GsT, in which all the linkages have Spphosphorus.

In some embodiments, a unimer is a “P-modification unimer”, e.g., allnucleotide units have the same modification at the linkage phosphorus.For instance, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO:106), in which all the internucleotidic linkages are phosphorothioatediester.

In some embodiments, a unimer is a “linkage unimer,” e.g., allnucleotide units have the same stereochemistry and the samemodifications at the linkage phosphorus. For instance,All-(Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO: 106), inwhich all the internucleotidic linkages are phosphorothioate diesterhaving Sp linkage phosphorus.

Gapmer: as used herein, the term “gapmer” refers to an oligonucleotidestrand characterized in that at least one internucleotidic phosphoruslinkage of the oligonucleotide strand is a phosphate diester linkage,for example such as those found in naturally occurring DNA or RNA. Insome embodiments, more than one internucleotidic phosphorus linkage ofthe oligonucleotide strand is a phosphate diester linkage such as thosefound in naturally occurring DNA or RNA. For instance, All-(Sp)-CAs1GsT,in which the internucleotidic linkage between C and A is a phosphatediester linkage.

Skipmer: as used herein, the term “skipmer” refers to a type of gapmerin which every other internucleotidic phosphorus linkage of theoligonucleotide strand is a phosphate diester linkage, for example suchas those found in naturally occurring DNA or RNA, and every otherinternucleotidic phosphorus linkage of the oligonucleotide strand is amodified internucleotidic linkage. For instance,All-(Sp)-AsTCs1GAs2TCs3G.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The methods and structures described herein relating to compounds andcompositions of the invention also apply to the pharmaceuticallyacceptable acid or base addition salts and all stereoisomeric forms ofthese compounds and compositions.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Chirally controlled oligonucleotide has significantly differentretention time on HPLC compared to the stereorandom oligonucleotide. A:crude chirally controlled oligonucleotide (Oligonucleotide 101); C: thecorresponding stereorandom oligonucleotide (Oligonucleotide 118).

FIG. 2. HPLC of chirally controlled oligonucleotides and stereorandomoligonucleotide. A: Oligonucleotide 101 (all-Rp); B: Oligonucleotide 102(all-Sp); and C: Oligonucleotide 118 (stereorandom).

FIG. 3. Tm of chirally controlled oligonucleotides and stereorandomoligonucleotide.

FIG. 4. Representative Data: Melting Curve Analysis of the target andendogenous control pairs yield single amplicons.

FIG. 5. Representative data and IC₅₀ curves for compounds.

FIG. 6. HPLC of crude (Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp,Rp, Rp, Sp, Rp, Rp, Sp, Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC]((RRS)₆-R (SEQ ID NO: 106), stereoblockmer and P-modification unimer(s-unimer)).

FIG. 7. HPLC of purified (Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp,Sp, Rp, Rp, Sp, Rp, Rp, Sp,Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] ((RRS)₆-R (SEQ ID NO:106), stereoblockmer and P-modification unimer (s-unimer)).

FIG. 8. LCMS of (Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp,Sp, Rp, Rp, Sp, Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] ((RRS)₆-R(SEQ ID NO: 106), stereoblockmer and P-modification unimer (s-unimer)).

FIG. 9. HPLC of crude (Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp,Sp, Rp, Rp, Sp, Rp, Rp, Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC](S-(RRS)₆ (SEQ ID NO: 106), stereoblockmer and P-modification unimer(s-unimer)).

FIG. 10. HPLC of purified (Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp,Rp, Sp, Rp, Rp, Sp, Rp, Rp,Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (S-(RRS)₆ (SEQ ID NO:106), stereoblockmer and P-modification unimer (s-unimer)).

FIG. 11. LCMS of (Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp,Rp, Rp, Sp, Rp, Rp, Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC](S-(RRS)₆ (SEQ ID NO: 106), stereoblockmer and P-modification unimer(s-unimer)).

FIG. 12. HPLC of crude (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp,Rp, Sp, Rp, Rp, Sp, Rp, Rp) d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC](RS-(RRS)₅-RR (SEQ ID NO: 106), stereoblockmer and P-modification unimer(s-unimer)).

FIG. 13. HPLC of purified (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp,Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp)d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (RS-(RRS)₅-RR (SEQ ID NO:106), stereoblockmer and P-modification unimer (s-unimer)).

FIG. 14. LCMS of (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp,Sp, Rp, Rp, Sp, Rp, Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC](RS-(RRS)₅-RR (SEQ ID NO: 106), stereoblockmer and P-modification unimer(s-unimer)).

FIG. 15. HPLC of crude (Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Rp,Rp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQ ID NO: 108)(3R-5S-3R, stereoblockmer and P-modification unimer (s1-unimer)).

FIG. 16. HPLC of purified (Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Rp,Rp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQ ID NO: 108)(3R-5S-3R, stereoblockmer and P-modification unimer (s1-unimer)).

FIG. 17. LCMS of (Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Rp,Rp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQ ID NO: 108)(3R-5S-3R, stereoblockmer and P-modification unimer (s1-unimer)).

FIG. 18. HPLC of crude All-(Rp)-d[Cs3As3Gs3T] (P-modification unimer(s3-unimer), stereounimer and linkage unimer).

FIG. 19. LCMS of All-(Rp)-d[Cs3As3Gs3T] (P-modification unimer(s3-unimer), stereounimer and linkage unimer).

FIG. 20. HPLC of crude All-(Rp)-d[Cs2As2Gs2T] (P-modification unimer(s2-unimer), stereounimer and linkage unimer).

FIG. 21. LCMS of All-(Rp)-d[Cs2As2Gs2T] (P-modification unimer(s2-unimer), stereounimer and linkage unimer).

FIG. 22. HPLC of crude All-(Sp)-d[Cs1AGs1T] (gapmer, stereoaltmer,P-modification altmer and linkage altmer).

FIG. 23. LCMS of All-(Sp)-d[Cs1AGs1T] (gapmer stereoaltmer,P-modification altmer and linkage altmer).

FIG. 24. Crude All-(Rp)-d[TsCs1AsT] (stereounimer, P-modification altmerand linkage altmer).

FIG. 25. LCMS of All-(Rp)-d[TsCs1AsT] (stereounimer, P-modificationaltmer and linkage altmer).

FIG. 26. Exemplary oligonucleotides (SEQ ID NOS 133 and 133,respectively, in order of appearance) described in WO2012/030683 andcontemplated for synthesis using methods of the present invention.

FIG. 27. Exemplary oligonucleotides (SEQ ID NOS 134-135, 135, 134, 136and 135, respectively, in order of appearance) described inWO2012/030683 and contemplated for synthesis using methods of thepresent invention.

FIG. 28. Exemplary oligonucleotides (SEQ ID NOS 135, 135, 135, 135, 135and 137, respectively, in order of appearance) described inWO2012/030683 and contemplated for synthesis using methods of thepresent invention.

FIG. 29. Exemplary oligonucleotides (SEQ ID NOS 136, 138, 136 and 137,respectively, in order of appearance) described in WO2012/030683 andcontemplated for synthesis using methods of the present invention.

FIG. 30. Exemplary oligonucleotides (SEQ ID NOS 134, 133 and 133,respectively, in order of appearance) described in WO2012/030683 andcontemplated for synthesis using methods of the present invention.

FIG. 31. Exemplary linkers described in WO2012/030683 for use in methodsof the present invention.

FIG. 32. Exemplary linkers described in WO2012/030683 for use in methodsof the present invention.

FIG. 33. Exemplary linkers described in WO2012/030683 for use in methodsof the present invention.

FIG. 34. Exemplary linkers described in WO2012/030683 for use in methodsof the present invention.

FIG. 35. RP-HPLC of crude DMT on oligonucleotide: ONT-75 (Panel A);ONT-80 (Panel B); ONT-77 (Panel C); ONT-81 (Panel D); ONT-87 (Panel E);ONT-88 (Panel F); ONT-89 (Panel G); ONT-82 (Panel H); ONT-84 (Panel I);ONT-85 (Panel J); ONT-86 (Panel K).

FIG. 36. RP-HPLC of purified DMT off oligonucleotide: ONT-75 (Panel A);ONT-80 (Panel B); ONT-77 (Panel C); ONT-81 (Panel D); ONT-87 (Panel E);ONT-88 (Panel F); ONT-89 (Panel G); ONT-82 (Panel H); ONT-84 (Panel I);ONT-85 (Panel J); ONT-86 (Panel K).

FIG. 37. Overlay of RP-HPLC traces of purified DMT off oligonucleotide:ONT-75, ONT-77, ONT-80, ONT-81, ONT-87, ONT-88, ONT-89, and ONT-41(Panel A); expanded view of overlay of ONT-75, ONT-77, ONT-80, ONT-81,ONT-87, ONT-88, ONT-89, and ONT-41 (Panel B).

FIG. 38. Overlay of RP-HPLC traces of purified DMT off oligonucleotide:ONT-82, ONT-84, ONT-85, ONT-86, and ONT-83 (Panel A); expanded view ofoverlay of ONT-82, ONT-84, ONT-85, ONT-86, and ONT-83 (Panel B).

FIG. 39. Tm overlay of chirally controlled oligonucleotides ONT-81,ONT-41, ONT-75, ONT-77, and ONT-80.

FIG. 40. Graphical representation of timecourse of serum humanapolipoprotein B protein levels relative to PBS after 5 mg/kgstereoisomer or mipomersen IP dosing in huApoB mice for ONT-41, ONT-75,ONT-80, ONT-77, and ONT-81. A downward arrow indicates dosing days.

FIG. 41. Graphical representation of timecourse of serum humanapolipoprotein B protein levels relative to PBS after 5 mg/kgstereoisomer or mipomersen IP dosing in huApoB mice for mipomersen,“full R” mipomersen, “full S” mipomersen, “RSR” mipomersen, and “SRS”mipomersen. A downward arrow indicates dosing days.

FIG. 42. Graphical representation of timecourse of serum humanapolipoprotein B protein levels relative to PBS after 10 mg/kgstereoisomer or mipomersen IP dosing in huApoB mice for mipomersen,“full R” mipomersen, “full S” mipomersen, “RSR” mipomersen, and “SRS”mipomersen. A downward arrow indicates dosing days.

FIG. 43. Graphical representation of timecourse of serum humanapolipoprotein B protein levels relative to PBS after 5 mg/kgstereoisomer or mipomersen IP dosing in huApoB mice for mipomersen,ONT-87, ONT-88, and ONT-89. A downward arrow indicates dosing days.

FIG. 44. Graphical representation of timecourse of serum humanapolipoprotein B protein levels relative to PBS after 10 mg/kgstereoisomer or mipomersen IP dosing in huApoB mice for ONT-87, ONT-88,and ONT-89. A downward arrow indicates dosing days.

FIG. 45. Graphical representation of % PCSK-9 mRNA remaining after Hep3Btreatment with siRNA duplex.

FIG. 46. Graphical representation of % PCSK-9 mRNA remaining after Hep3Btreatment with siRNA duplex curve fit.

FIG. 47. Graphical representation of % PCSK-9 mRNA remaining after HeLatreatment with siRNA duplex.

FIG. 48. Graphical representation of % PCSK-9 mRNA remaining after HeLatreatment with siRNA duplex curve fit.

FIG. 49. Graphical representation of % PCSK-9 mRNA remaining after HeLatreatment with siRNA duplex containing 3 Phosphorothiate stereo-centers.

FIG. 50. Graphical representation of % PCSK-9 mRNA remaining after HeLatreatment with siRNA duplex containing 3 Phosphorothiate stereo-centerscurve fit.

FIG. 51. Overlay of RP-HPLC traces of purified DMT off oligonucleotide:ONT-108, ONT-109, and ONT-114.

FIG. 52. Overlay of RP-HPLC traces of purified DMT off oligonucleotide:ONT-106, ONT-107, and ONT-114.

FIG. 53. Graphical representation of timecourse of serum humanapolipoprotein B protein levels relative to PBS after 10 mg/kgstereoisomer or mipomersen IP dosing in huApoB mice. A downward arrowindicates dosing days.

FIG. 54. Graphical representation of timecourse of serum humanapolipoprotein B protein levels relative to PBS after multiple IP dosesof 5 mg/kg stereoisomer or mipomersen in huApoB mice (n=3-4). A downwardarrow indicates dosing days.

FIG. 55. Day 17 serum human apolipoprotein B protein levels relative toPBS after 10 mg/kg stereoisomer (ONT-87, ONT-88 or ONT-89) or mipomersenIP dosing in huApoB mice.

FIG. 56. Day 24 Serum Human Apolipoprotein B Protein Levels Relative toPBS After 10 mg/kg Stereoisomer (ONT-87, ONT-88 or ONT-89) or MipomersenIP Dosing in huApoB Mice.

FIG. 57. Serum Human Apolipoprotein B Protein Levels Relative to PBSAfter 10 mg/kg Stereoisomer (ONT-41, ONT-87, ONT-88 or ONT-89) Dosing inhuApoB Mice.

FIG. 58. Serum Human Apolipoprotein B Protein Levels Relative to PBSAfter 10 mg/kg Stereoisomer (ONT-87, ONT-88 or ONT-89) Dosing in huApoBMice.

FIG. 59. Plot of IEX-HPLC quantification analysis of svPDE digestionstudy for oligonucleotides ONT-75, ONT-77, ONT-80, ONT-81, ONT-87,ONT-88, ONT-89 and ONT-41.

FIG. 60. IEX-HPLC of enzymatic digestion study using nP1 foroligonucleotide ONT-75 (All(Rp))-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsmCsTsTs5mCsGs 5mCsAs5mCs5mC (SEQ IDNO: 106).

FIG. 61. IEX-HPLC of enzymatic digestion study using nP1 foroligonucleotide ONT-77 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,Sp, Sp, Sp, Rp, Rp, Rp,Rp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ IDNO: 106) (5R-10S-4R).

FIG. 62. IEX-HPLC of enzymatic digestion study using nP1 foroligonucleotide ONT-80 (All(Sp))-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCs Gs5mCsAs5mCs5mC (SEQ IDNO: 106).

FIG. 63. IEX-HPLC of enzymatic digestion study using nP1 foroligonucleotide ONT-81 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp,Rp, Rp, Rp, Sp, Sp, Sp,Sp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ IDNO: 106) (5S-10R-4S).

FIG. 64. IEX-HPLC of enzymatic digestion study using nP1 foroligonucleotide ONT-87 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp, Rp, Rp, Rp, Rp, Rp,Rp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ IDNO: 106) (5R-(SSR)₃-5R).

FIG. 65. IEX-HPLC of enzymatic digestion study using nP1 foroligonucleotide ONT-88 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp,Rp, Sp, Sp, Sp, Sp, Sp,Sp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ IDNO: 106) (5S-(RRS)₃-5SS).

FIG. 66. IEX-HPLC of enzymatic digestion study using nP1 foroligonucleotide ONT-89 (Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp, Rp, Sp, Rp, Sp, Rp,Sp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ IDNO: 106) ((SR)₉S).

FIG. 67. IEX-HPLC of enzymatic digestion study using nP1 foroligonucleotide ONT-41 (diastereomixture)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ ID NO: 106).

FIG. 68. Comparison of stability of chirally pure oligonucleotidesONT-75 and ONT-77 with the stereorandom “parental” oligonucleotideONT-41 (Mipomersen) in preincubated rat whole liver homogenate.

FIG. 69. UPLC profile in producing oligonucleotide derivative using themonomer of 13b.

FIG. 70. UPLC profile in producing oligonucleotide derivative using themonomer of 27.

FIG. 71. Mouse Apolipoprotein B/GAPDH mRNA Levels Relative to Mock andUntreated Controls after Transfection of Primary Mouse Hepatocytes withStereoisomer (ONT-82, ONT-83, ONT-84, ONT-85 or ONT-86).

FIG. 72. Mouse Apolipoprotein B/GAPDH mRNA Levels Relative to Mock andUntreated Controls after Transfection of Primary Mouse Hepatocytes withStereoisomer (ONT-83, ONT-84, ONT-85 or ONT-86).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Synthetic oligonucleotides provide useful molecular tools in a widevariety of applications. For example, oligonucleotides are useful intherapeutic, diagnostic, research, and new nanomaterials applications.The use of naturally occurring nucleic acids (e.g., unmodified DNA orRNA) is limited, for example, by their susceptibility to endo- andexo-nucleases. As such, various synthetic counterparts have beendeveloped to circumvent these shortcomings. These include syntheticoligonucleotides that contain backbone modifications, which render thesemolecules less susceptible to degradation. From a structural point ofview, such modifications to internucleotide phosphate linkages introducechirality. It has become clear that certain properties ofoligonucleotides may be affected by the configurations of the phosphorusatoms that form the backbone of the oligonucleotides. For example, invitro studies have shown that the properties of antisense nucleotidessuch as binding affinity, sequence specific binding to the complementaryRNA, stability to nucleases are affected by, inter alia, chirality ofthe backbone (e.g., the configurations of the phosphorus atoms). Thus,the present invention encompasses the recognition that there is a needfor chirally controlled oligonucleotides which comprise phosphorusatom-modified nucleic acids, as well as related compositions andmethods. In some embodiments, the present invention provides chirallycontrolled oligonucleotides that are structurally optimized to exhibitcertain desirable characteristics, such as, e.g., increased stabilityand improved efficacy for in vitro and/or in vivo applications.

Oligonucleotides in which one or two of the two nonbridging oxygen atomsof the internucleotidic phosphates is replaced by a different type ofatom or substituent are known to be useful as therapeutic agents andprobes to elucidate enzymatic reaction mechanisms. However, sucholigonucleotides often exhibit undesirable properties (e.g.,susceptibility to degradation by nucleases, poor cell membranepermeability) that prohibit their use in numerous applications. Thus,various types of chemical modifications have been developed in anattempt to improve their properties and/or impart new functionality.

Modified Oligonucleotide Structures

As noted above, in light of the usefulness of oligonucleotidecompositions in various applications and indications, those skilled inthe art have endeavored to develop modifications of oligonucleotidestructures that may have preferred or desirable characteristics orattributes as compared with naturally-occurring oligonucleotidemolecules, for example as used in particular applications andindications. Exemplary such modifications are described below.

WO2010/141471 (herein “Traversa I”) teaches the modification ofdifferent types of nucleic acid constructs modified to have a reducednet polyanionic charge. WO2010/039543 (herein “Travera II”) teachescompositions and methods for making neutral polynucleotides (NNs) withreduced polyanionic charge. WO2008/008476 (herein, “Traversa III”)describes the synthesis of SATE (Imbach-type) phosphate prodrugs.Traversa I, II, and III do not teach chirally controlledoligonucleotides, compositions thereof, and methods of making and usingthe same, as described by the present invention.

WO2010/072831 (herein “Girindus et al.”) also teaches the modificationof oligonucleotides. In particular, Girindus et al. teaches the use ofsulfurization reagents to generate phosphorothioate triesters asprodrugs. Girindus et al. does not teach chirally controlledoligonucleotides, compositions thereof, and methods of making and usingthe same, as described by the present invention.

Similarly, WO2004/085454 (herein “Avecia I”) teaches the preparation ofphosphorothioate oligonucleotides through, e.g., transient silylation ofpoly-H-phosphonate diesters. WO2001/027126 (herein “Avecia II”) teachesprocesses for the solid phase synthesis of phosphotriesteroligonucleotides by coupling H-phosphonate monomers to a solid supported5′-hydroxyl oligonucleotide and further sulfurization of the resultingH-phosphonate diester into a phosphorothioate triester. The disclosureof WO2001/064702 (herein “Avecia III”) is similar to Avecia II andfurther describes solid-phase synthesis on different solid supports.Avecia I, II, and III do not teach chirally controlled oligonucleotides,compositions thereof, and methods of making and using the same, asdescribed by the present invention.

WO1997/006183 (herein “Chiron”) teaches oligonucleotides with cationicinternucleotide linkages comprising asymmetric phosphorus, such asstereopure amidates. Chiron teaches stereopure oligonucleotides obtainedvia crystallization of a mixture of diastereomers or via resolutionusing, e.g., column chromatography. Chiron does not teach chirallycontrolled oligonucleotides, compositions thereof, and methods of makingand using the same, as described by the present invention.

WO2009/146123 (herein “Spring Bank I”) teaches compositions and methodsfor treating viral infections using substituted phosphateoligonucleotides and phosphorothioate triesters. WO2007/070598 (herein“Spring Bank II”) teaches phosphotriester prodrugs as antiviral nucleicacids and teaches the synthesis of phosphorothioate prodrugs. SpringBank I and II do not teach chirally controlled oligonucleotides,compositions thereof, and methods of making and using the same, asdescribed by the present invention.

EP0779893 (herein “Hybridon”) teaches lipophilic prodrugs for theincreased cellular uptake of antisense oligonucleotides and observesthat Rp and Sp phosphorothioates and phosphorothioate triester dimerscan have different enzymatic stability properties. Hybridon does notteach chirally controlled oligonucleotides, compositions thereof, andmethods of making and using the same, as described by the presentinvention.

WO1997/047637 (herein “Imbach I”) teaches generally the Imbach “SATE”(S-acyl thioethyl) prodrug oligonucleotide compositions and methods.Imbach I describes, for example, bioreversible phosphotriester prodrugsand the preparation of certain prodrug oligonucleotides usingpost-synthetic alkylation or prodrug-group-containing phosphoramidites.U.S. Pat. No. 6,124,445 (herein “Imbach II”) teaches modified antisenseand chimeric prodrug oligonucleotides. Imbach I and II do not teachchirally controlled oligonucleotides, compositions thereof, and methodsof making and using the same, as described by the present invention.

WO2006/065751 (herein “Beaucage”) teaches CpG oligonucleotidephosphorothioate prodrugs that comprise thermolabile substituents (whichsubstituents are introduced via a phosphoramidite monomer), andapplications thereof. Beaucage does not teach chirally controlledoligonucleotides, compositions thereof, and methods of making and usingthe same, as described by the present invention.

Takeshi Wada et al. developed novel methods for the stereo-controlledsynthesis of P-chiral nucleic acids using amidite chiral auxiliaries(JP4348077, WO2005/014609, WO2005/092909, and WO2010/064146,cumulatively referred to herein as “Wada I”). In particular,WO2010/064146 (referred to herein as “Wada II”) discloses methods forsynthesizing phosphorus atom-modified nucleic acids wherein thestereochemical configuration at phosphorus is controlled. However, themethods of Wada II are limited in that they do not provide forindividual P-modification of each chiral linkage phosphorus in acontrolled and designed manner. That is, the methods for P-modifiedlinkages of Wada II provide for the generation of a condensedintermediate poly H-phosphonate oligonucleotide strand that, once builtto a desired length, is mass modified at the linkage phosphorus toprovide, e.g., a desired phosphorothioate diester, phosphoramidate orboranophosphate or other such phosphorus atom-modified nucleic acids(referred to as Route B in the document—Scheme 6, page 36). Furthermore,the H-phosphonate oligonucleotide strands of Wada II are of shorterlengths (e.g., dimer trimer, or tetramer). Combined with the fact thatthere is no capping step in route B, which generally presents low crudepurity as a result of the accumulation of “n−1”-type byproducts, theWada II route contains limitations in regards of the synthesis of longeroligonucleotides. While Wada II contemplates generally that a particularoligonucleotide could be envisaged to contain different modifications ateach linkage phosphorus, Wada II does not describe or suggest methodsfor controlled iterative installation of such modifications, as aredescribed herein. To the extent that Wada II depicts a synthetic cyclethat does not require an H-phosphonate intermediate oligonucleotide tobe completely assembled prior to modification at the linkage phosphorus(therein referred to as Route A, page 35, Scheme 5, “Synthesis of anucleic acid comprising a chiral X-phosphonate moiety of Formula 1 viaRoute A”), this general disclosure does not teach certain key steps thatare required to install certain P-modifications, as provided by thepresent invention, and especially not with any degree of efficiency andversatility such that this cycle would be useful in the synthesis ofchirally controlled P-modified oligonucleotides, and especiallyoligonucleotides of longer lengths.

At least one such inefficiency of Wada II is noted by Wada et al. inWO2012/039448 (herein “Wada III”). Wada III teaches novel chiralauxiliaries for use in Wada II methods to produce H-phosphonateoligonucleotides that, once built, can be subsequently modified toprovide, inter alia, phosphorothioates and the like. Wada et al. observein Wada III that the four types of chiral auxiliaries disclosed in WadaII formed strong bonds with phosphorus at the linkage phosphorus andthus did not allow for efficient removal. Wada III notes that removal ofthe Wada II chiral auxiliaries required harsh conditions, whichconditions were prone to compromising the integrity of the productoligonucleotide. Wada III observes that this is especially problematicwhen synthesizing long chain oligonucleotides for at least the reasonthat as the degradation reaction(s) proceed, additional byproducts aregenerated that can further react with and degrade the productoligonucleotide. Wada III therefore provides chiral auxiliaries that canbe more efficiently cleaved from the oligonucleotide under mild acidicconditions by way of an S_(N)1 mechanism releasing the H-phosphonateinternucleotide linkage (route B), or under relatively mild basicconditions, by a β-elimation pathway.

One of skill in the chemical and synthetic arts will immediatelyappreciate the complexities associated with generating chirallycontrolled oligonucleotides such as those provided by the presentinvention. For instance, in order to synthesize and isolate a chirallycontrolled oligonucleotide, conditions for each monomer addition must bedesigned such that (1) the chemistry is compatible with every portion ofthe growing oligonucleotide; (2) the byproducts generated during eachmonomer addition do not compromise the structural and stereochemicalintegrity of the growing oligonucleotide; and (3) the crude finalproduct composition is a composition which allows for isolation of thedesired chirally controlled oligonucleotide product.

Oligonucleotide phosphorothioates have shown therapeutic potential(Stein et al., Science (1993), 261:1004-12; Agrawal et al., AntisenceRes. and Dev. (1992), 2:261-66; Bayever et al., Antisense Res. and Dev.(1993), 3:383-390). Oligonucleotide phosphorothioates prepared withoutregard to the stereochemistry of the phosphorothioate exist as a mixtureof 2^(n) diastereomers, where n is the number of internucleotidephosphorothioates linkages. The chemical and biological properties ofthese diastereomeric phosphorothioates can be distinct. For example,Wada et al (Nucleic Acids Symposium Series No. 51 p. 119-120;doi:10.1093/nass/nrm060) found that stereodefined-(Rp)-(Ups)9U/(Ap)9Aduplex showed a higher Tm value than that of natural-(Up)9U/(Ap)9A andstereodefined-(Sp)-(Ups)9U did not form a duplex. In another example, ina study by Tang et al., (Nucleosides Nucleotides (1995), 14:985-990)stereopure Rp-oligodeoxyribonucleoside phosphorothioates were found topossess lower stability to nucleases endogenous to human serum that theparent oligodeoxyribonucleoside phosphorothioates with undefinedphosphorus chirality.

Chirally Controlled Oligonucleotides and Chirally ControlledOligonucleotide Compositions

The present invention provides chirally controlled oligonucleotides, andchirally controlled oligonucleotide compositions which are of high crudepurity and of high diastereomeric purity. In some embodiments, thepresent invention provides chirally controlled oligonucleotides, andchirally controlled oligonucleotide compositions which are of high crudepurity. In some embodiments, the present invention provides chirallycontrolled oligonucleotides, and chirally controlled oligonucleotidecompositions which are of high diastereomeric purity.

In some embodiments, the present invention provides chirally controlledcompositions comprising a plurality of oligonucleotides of at least onetype, wherein each type is defined by: 1) base sequence; 2) pattern ofbackbone linkages; 3) pattern of backbone chiral centers; and 4) patternof backbone P-modifications.

In some embodiments, the present invention provides chirally controlledcompositions comprising a plurality of oligonucleotides of the sametype, wherein each type is defined by: 1) base sequence; 2) pattern ofbackbone linkages; 3) pattern of backbone chiral centers; and 4) patternof backbone P-modifications. In some embodiments, the present inventionprovides chirally controlled compositions comprising a plurality ofoligonucleotides of two or more types, wherein each type is definedby: 1) base sequence; 2) pattern of backbone linkages; 3) pattern ofbackbone chiral centers; and 4) pattern of backbone P-modifications.

In some embodiments, the present invention provides oligonucleotidescomprising one or more diastereomerically pure internucleotidic linkageswith respect to the chiral linkage phosphorus. In some embodiments, thepresent invention provides oligonucleotides comprising one or morediastereomerically pure internucleotidic linkages having the structureof formula I. In some embodiments, the present invention providesoligonucleotides comprising one or more diastereomerically pureinternucleotidic linkages with respect to the chiral linkage phosphorus,and one or more phosphate diester linkages. In some embodiments, thepresent invention provides oligonucleotides comprising one or morediastereomerically pure internucleotidic linkages having the structureof formula I, and one or more phosphate diester linkages. In someembodiments, the present invention provides oligonucleotides comprisingone or more diastereomerically pure internucleotidic linkages having thestructure of formula I-c, and one or more phosphate diester linkages. Insome embodiments, such oligonucleotides are prepared by usingstereoselective oligonucleotide synthesis, as described in thisapplication, to form pre-designed diastereomerically pureinternucleotidic linkages with respect to the chiral linkage phosphorus.For instance, in one exemplary oligonucleotide of (Rp/Sp, Rp/Sp, Rp/Sp,Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGs1Cs1As1CsC] (SEQ ID NO: 106), thefirst three internucleotidic linkages are constructed using traditionaloligonucleotide synthesis method, and the diastereomerically pureinternucleotidic linkages are constructed with stereochemical control asdescribed in this application. Exemplary internucleotidic linkages,including those having structures of formula I, are further describedbelow. In some embodiments, such oligonucleotides comprise a sequencefurther described in the application, including but not limited to thosedescribed in Tables 2 and 4, and Appendices A, B and C.

In some embodiments, a provided oligonucleotide comprises a combinationof stereopure and stereorandom internucleotidic linkages with respect tochirality at the linkage phosphorus. For instance, in some embodimentsit is desirable to have a block of one or more stereodefinedinternucleotidic linkages within an oligonucleotide that is otherwisestereorandom with respect to chirality at the linkage phosphorus. Insome embodiments, it is desirable to have a block of one or moreinternucleotidic linkages that are stereorandom within anoligonucleotide that is otherwise stereodefined with respect tochirality at the linkage phosphorus.

In some embodiments, at least one nucleotide unit of a providedoligonucleotide is installed using stereoselective oligonucleotidesynthesis, as described in this application, to form a pre-designeddiastereomerically pure internucleotidic linkage with respect to thechiral linkage phosphorus. In some embodiments, at least two nucleotideunits of a provided oligonucleotide are installed using stereoselectiveoligonucleotide synthesis, as described in this application, to form atleast two pre-designed diastereomerically pure internucleotidic linkageswith respect to the chiral linkage phosphorus. In some embodiments, atleast three nucleotide units of a provided oligonucleotide are installedusing stereoselective oligonucleotide synthesis, as described in thisapplication, to form at least three pre-designed diastereomerically pureinternucleotidic linkages with respect to the chiral linkage phosphorus.In some embodiments, the at least one, two, or three pre-designeddiastereomerically pure internucleotidic linkages are adjacent to oneanother. In some embodiments, the at least one, two, or threepre-designed diastereomerically pure internucleotidic linkages are notadjacent to one another.

In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, or 50% of nucleotide units of a provided oligonucleotide areinstalled using stereoselective oligonucleotide synthesis, as describedin this application, to form a pre-designed diastereomerically pureinternucleotidic linkage with respect to the chiral linkage phosphorus.As described herein, in some embodiments the at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of nucleotide units occur inone or more blocks to provide a blockmer. In some embodiments, the atleast about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% ofnucleotide units occur in a an alternating pattern to provide an altmer.One of skill in the relevant arts will recognize that any desirablepattern can be achieved using methods of the present invention and arecontemplated herein.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry and/or different P-modifications relative to oneanother. In certain embodiments, the present invention provides achirally controlled oligonucleotide, wherein at least two individualinternucleotidic linkages within the oligonucleotide have differentP-modifications relative to one another. In certain embodiments, thepresent invention provides a chirally controlled oligonucleotide,wherein at least two of the individual internucleotidic linkages withinthe oligonucleotide have different P-modifications relative to oneanother, and wherein the chirally controlled oligonucleotide comprisesat least one phosphate diester internucleotidic linkage. In certainembodiments, the present invention provides a chirally controlledoligonucleotide, wherein at least two of the individual internucleotidiclinkages within the oligonucleotide have different P-modificationsrelative to one another, and wherein the chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate diesterinternucleotidic linkage. In certain embodiments, the present inventionprovides a chirally controlled oligonucleotide, wherein at least two ofthe individual internucleotidic linkages within the oligonucleotide havedifferent P-modifications relative to one another, and wherein thechirally controlled oligonucleotide comprises at least onephosphorothioate triester internucleotidic linkage. In certainembodiments, the present invention provides a chirally controlledoligonucleotide, wherein at least two of the individual internucleotidiclinkages within the oligonucleotide have different P-modificationsrelative to one another, and wherein the chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate triesterinternucleotidic linkage.

In certain embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising one or more modifiedinternucleotidic linkages independently having the structure of formulaI:

wherein each variable is as defined and described below. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising one or more modified internucleotidiclinkages of formula I, and wherein individual internucleotidic linkagesof formula I within the oligonucleotide have different P-modificationsrelative to one another. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide comprising one or moremodified internucleotidic linkages of formula I, and wherein individualinternucleotidic linkages of formula I within the oligonucleotide havedifferent —X-L-R¹ relative to one another. In some embodiments, thepresent invention provides a chirally controlled oligonucleotidecomprising one or more modified internucleotidic linkages of formula Iand wherein individual internucleotidic linkages of formula I within theoligonucleotide have different X relative to one another. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising one or more modified internucleotidiclinkages of formula I, and wherein individual internucleotidic linkagesof formula I within the oligonucleotide have different -L-R¹ relative toone another.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry and/or different P-modifications relative to oneanother. In some embodiments, the present invention provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry relative to one another, and wherein at least a portionof the structure of the chirally controlled oligonucleotide ischaracterized by a repeating pattern of alternating stereochemistry.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentP-modifications relative to one another, in that they have different Xatoms in their —XLR¹ moieties, and/or in that they have different Lgroups in their —XLR¹ moieties, and/or that they have different R¹ atomsin their —XLR¹ moieties.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry and/or different P-modifications relative to one anotherand the oligonucleotide has a structure represented by the followingformula:

[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny]

wherein:each R^(B) independently represents a block of nucleotide units havingthe R configuration at the linkage phosphorus;each S^(B) independently represents a block of nucleotide units havingthe S configuration at the linkage phosphorus;each of n1-ny is zero or an integer, with the requirement that at leastone odd n and at least one even n must be non-zero so that theoligonucleotide includes at least two individual internucleotidiclinkages with different stereochemistry relative to one another; andwherein the sum of n1-ny is between 2 and 200, and in some embodimentsis between a lower limit selected from the group consisting of 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or more and an upper limit selected from the group consisting of5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200, the upperlimit being larger than the lower limit.

In some such embodiments, each n has the same value; in someembodiments, each even n has the same value as each other even n; insome embodiments, each odd n has the same value each other odd n; insome embodiments, at least two even ns have different values from oneanother; in some embodiments, at least two odd ns have different valuesfrom one another.

In some embodiments, at least two adjacent ns are equal to one another,so that a provided oligonucleotide includes adjacent blocks of Sstereochemistry linkages and R stereochemistry linkages of equallengths. In some embodiments, provided oligonucleotides includerepeating blocks of S and R stereochemistry linkages of equal lengths.In some embodiments, provided oligonucleotides include repeating blocksof S and R stereochemistry linkages, where at least two such blocks areof different lengths from one another; in some such embodiments each Sstereochemistry block is of the same length, and is of a differentlength from each R stereochemistry length, which may optionally be ofthe same length as one another.

In some embodiments, at least two skip-adjacent ns are equal to oneanother, so that a provided oligonucleotide includes at least two blocksof linkages of a first stereochemistry that are equal in length to oneanother and are separated by a block of linkages of the otherstereochemistry, which separating block may be of the same length or adifferent length from the blocks of first steroechemistry.

In some embodiments, ns associated with linkage blocks at the ends of aprovided oligonucleotide are of the same length. In some embodiments,provided oligonucleotides have terminal blocks of the same linkagestereochemistry. In some such embodiments, the terminal blocks areseparated from one another by a middle block of the other linkagestereochemistry.

In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a stereoblockmer.In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a stereoskipmer.In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a stereoaltmer.In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a gapmer.

In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is of any of theabove described patterns and further comprises patterns ofP-modifications. For instance, in some embodiments, a providedoligonucleotide of formula [S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . .S^(B)nxR^(B)ny] and is a stereoskipmer and P-modification skipmer. Insome embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] and is astereoblockmer and P-modification altmer. In some embodiments, aprovided oligonucleotide of formula [S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . .S^(B)nxR^(B)ny] and is a stereoaltmer and P-modification blockmer.

In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a chirallycontrolled oligonucleotide comprising one or more modifiedinternucleotidic linkages independently having the structure of formulaI:

wherein:

-   P* is an asymmetric phosphorus atom and is either Rp or Sp;-   W is O, S or Se;-   each of X, Y and Z is independently —O—, —S—, —N(-L-R¹)—, or L;-   L is a covalent bond or an optionally substituted, linear or    branched C₁-C₁₀ alkylene, wherein one or more methylene units of L    are optionally and independently replaced by an optionally    substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,    —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,    —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or    —C(O)O—;-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆    alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—,    —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,    —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,    —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ on the same nitrogen are taken together with their        intervening atoms to form an optionally substituted heterocyclic        or heteroaryl ring, or    -   two R′ on the same carbon are taken together with their        intervening atoms to form an optionally substituted aryl,        carbocyclic, heterocyclic, or heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, or    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl; and-   each

independently represents a connection to a nucleoside.

In some embodiments, a chirally controlled oligonucleotide comprises oneor more modified internucleotidic phosphorus linkages. In someembodiments, a chirally controlled oligonucleotide comprises, e.g., aphosphorothioate or a phosphorothioate triester linkage. In someembodiments, a chirally controlled oligonucleotide comprises aphosphorothioate triester linkage. In some embodiments, a chirallycontrolled oligonucleotide comprises at least two phosphorothioatetriester linkages. In some embodiments, a chirally controlledoligonucleotide comprises at least three phosphorothioate triesterlinkages. In some embodiments, a chirally controlled oligonucleotidecomprises at least four phosphorothioate triester linkages. In someembodiments, a chirally controlled oligonucleotide comprises at leastfive phosphorothioate triester linkages. Exemplary such modifiedinternucleotidic phosphorus linkages are described further herein.

In some embodiments, a chirally controlled oligonucleotide comprisesdifferent internucleotidic phosphorus linkages. In some embodiments, achirally controlled oligonucleotide comprises at least one phosphatediester internucleotidic linkage and at least one modifiedinternucleotidic linkage. In some embodiments, a chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate triesterlinkage. In some embodiments, a chirally controlled oligonucleotidecomprises at least one phosphate diester internucleotidic linkage and atleast two phosphorothioate triester linkages. In some embodiments, achirally controlled oligonucleotide comprises at least one phosphatediester internucleotidic linkage and at least three phosphorothioatetriester linkages. In some embodiments, a chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least four phosphorothioate triesterlinkages. In some embodiments, a chirally controlled oligonucleotidecomprises at least one phosphate diester internucleotidic linkage and atleast five phosphorothioate triester linkages. Exemplary such modifiedinternucleotidic phosphorus linkages are described further herein.

In some embodiments, a phosphorothioate triester linkage comprises achiral auxiliary, which, for example, is used to control thestereoselectivity of a reaction. In some embodiments, a phosphorothioatetriester linkage does not comprise a chiral auxiliary. In someembodiments, a phosphorothioate triester linkage is intentionallymaintained until and/or during the administration to a subject.

In some embodiments, a chirally controlled oligonucleotide is linked toa solid support. In some embodiments, a chirally controlledoligonucleotide is cleaved from a solid support.

In some embodiments, a chirally controlled oligonucleotide comprises atleast one phosphate diester internucleotidic linkage and at least twoconsecutive modified internucleotidic linkages. In some embodiments, achirally controlled oligonucleotide comprises at least one phosphatediester internucleotidic linkage and at least two consecutivephosphorothioate triester internucleotidic linkages.

In some embodiments, a chirally controlled oligonucleotide is ablockmer. In some embodiments, a chirally controlled oligonucleotide isa stereoblockmer. In some embodiments, a chirally controlledoligonucleotide is a P-modification blockmer. In some embodiments, achirally controlled oligonucleotide is a linkage blockmer.

In some embodiments, a chirally controlled oligonucleotide is an altmer.In some embodiments, a chirally controlled oligonucleotide is astereoaltmer. In some embodiments, a chirally controlled oligonucleotideis a P-modification altmer. In some embodiments, a chirally controlledoligonucleotide is a linkage altmer.

In some embodiments, a chirally controlled oligonucleotide is a unimer.In some embodiments, a chirally controlled oligonucleotide is astereounimer. In some embodiments, a chirally controlled oligonucleotideis a P-modification unimer. In some embodiments, a chirally controlledoligonucleotide is a linkage unimer.

In some embodiments, a chirally controlled oligonucleotide is a gapmer.

In some embodiments, a chirally controlled oligonucleotide is a skipmer.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising one or more modifiedinternucleotidic linkages independently having the structure of formulaI:

wherein:

-   P* is an asymmetric phosphorus atom and is either Rp or Sp;-   W is O, S or Se;-   each of X, Y and Z is independently —O—, —S—, —N(-L-R¹)—, or L;-   L is a covalent bond or an optionally substituted, linear or    branched C₁-C₁₀ alkylene, wherein one or more methylene units of L    are optionally and independently replaced by an optionally    substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,    —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,    —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or    —C(O)O—;-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆    alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—,    —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,    —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,    —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ on the same nitrogen are taken together with their        intervening atoms to form an optionally substituted heterocyclic        or heteroaryl ring, or    -   two R′ on the same carbon are taken together with their        intervening atoms to form an optionally substituted aryl,        carbocyclic, heterocyclic, or heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, or    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl; and-   each    independently represents a connection to a nucleoside.

As defined generally above and herein, P* is an asymmetric phosphorusatom and is either Rp or Sp. In some embodiments, P* is Rp. In otherembodiments, P* is Sp. In some embodiments, an oligonucleotide comprisesone or more internucleotidic linkages of formula I wherein each P* isindependently Rp or Sp. In some embodiments, an oligonucleotidecomprises one or more internucleotidic linkages of formula I whereineach P* is Rp. In some embodiments, an oligonucleotide comprises one ormore internucleotidic linkages of formula I wherein each P* is Sp. Insome embodiments, an oligonucleotide comprises at least oneinternucleotidic linkage of formula I wherein P* is Rp. In someembodiments, an oligonucleotide comprises at least one internucleotidiclinkage of formula I wherein P* is Sp. In some embodiments, anoligonucleotide comprises at least one internucleotidic linkage offormula I wherein P* is Rp, and at least one internucleotidic linkage offormula I wherein P* is Sp.

As defined generally above and herein, W is O, S, or Se. In someembodiments, W is O. In some embodiments, W is S. In some embodiments, Wis Se. In some embodiments, an oligonucleotide comprises at least oneinternucleotidic linkage of formula I wherein W is O. In someembodiments, an oligonucleotide comprises at least one internucleotidiclinkage of formula I wherein W is S. In some embodiments, anoligonucleotide comprises at least one internucleotidic linkage offormula I wherein W is Se.

As defined generally above and herein, each R is independently hydrogen,or an optionally substituted group selected from C₁-C₆ aliphatic,phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R is hydrogen. In some embodiments, R is anoptionally substituted group selected from C₁-C₆ aliphatic, phenyl,carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R is an optionally substituted C₁-C₆ aliphatic. Insome embodiments, R is an optionally substituted C₁-C₆ alkyl. In someembodiments, R is optionally substituted, linear or branched hexyl. Insome embodiments, R is optionally substituted, linear or branchedpentyl. In some embodiments, R is optionally substituted, linear orbranched butyl. In some embodiments, R is optionally substituted, linearor branched propyl. In some embodiments, R is optionally substitutedethyl. In some embodiments, R is optionally substituted methyl.

In some embodiments, R is optionally substituted phenyl. In someembodiments, R is substituted phenyl. In some embodiments, R is phenyl.

In some embodiments, R is optionally substituted carbocyclyl. In someembodiments, R is optionally substituted C₃-C₁₀ carbocyclyl. In someembodiments, R is optionally substituted monocyclic carbocyclyl. In someembodiments, R is optionally substituted cycloheptyl. In someembodiments, R is optionally substituted cyclohexyl. In someembodiments, R is optionally substituted cyclopentyl. In someembodiments, R is optionally substituted cyclobutyl. In someembodiments, R is an optionally substituted cyclopropyl. In someembodiments, R is optionally substituted bicyclic carbocyclyl.

In some embodiments, R is an optionally substituted aryl. In someembodiments, R is an optionally substituted bicyclic aryl ring.

In some embodiments, R is an optionally substituted heteroaryl. In someembodiments, R is an optionally substituted 5-6 membered monocyclicheteroaryl ring having 1-3 heteroatoms independently selected fromnitrogen, sulfur, or oxygen. In some embodiments, R is a substituted 5-6membered monocyclic heteroaryl ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anunsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, sulfur, or oxygen.

In some embodiments, R is an optionally substituted 5 memberedmonocyclic heteroaryl ring having 1-3 heteroatoms independently selectedfrom nitrogen, oxygen or sulfur. In some embodiments, R is an optionallysubstituted 6 membered monocyclic heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted 5-memberedmonocyclic heteroaryl ring having 1 heteroatom selected from nitrogen,oxygen, or sulfur. In some embodiments, R is selected from pyrrolyl,furanyl, or thienyl.

In some embodiments, R is an optionally substituted 5-memberedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, R is an optionallysubstituted 5-membered heteroaryl ring having 1 nitrogen atom, and anadditional heteroatom selected from sulfur or oxygen. Exemplary R groupsinclude optionally substituted pyrazolyl, imidazolyl, thiazolyl,isothiazolyl, oxazolyl or isoxazolyl.

In some embodiments, R is a 6-membered heteroaryl ring having 1-3nitrogen atoms. In other embodiments, R is an optionally substituted6-membered heteroaryl ring having 1-2 nitrogen atoms. In someembodiments, R is an optionally substituted 6-membered heteroaryl ringhaving 2 nitrogen atoms. In certain embodiments, R is an optionallysubstituted 6-membered heteroaryl ring having 1 nitrogen. Exemplary Rgroups include optionally substituted pyridinyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazinyl, or tetrazinyl.

In certain embodiments, R is an optionally substituted 8-10 memberedbicyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In certain embodiments, R is an optionally substituted 5,6-fusedheteroaryl ring having 1 heteroatom independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is an optionallysubstituted indolyl. In some embodiments, R is an optionally substitutedazabicyclo[3.2.1]octanyl. In certain embodiments, R is an optionallysubstituted 5,6-fused heteroaryl ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted azaindolyl. In some embodiments, R is anoptionally substituted benzimidazolyl. In some embodiments, R is anoptionally substituted benzothiazolyl. In some embodiments, R is anoptionally substituted benzoxazolyl. In some embodiments, R is anoptionally substituted indazolyl. In certain embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R is an optionally substituted 6,6-fusedheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is an optionallysubstituted 6,6-fused heteroaryl ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R is an optionally substituted 6,6-fused heteroaryl ringhaving 1 heteroatom independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R is an optionally substituted quinolinyl.In some embodiments, R is an optionally substituted isoquinolinyl.According to one aspect, R is an optionally substituted 6,6-fusedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is a quinazoline ora quinoxaline.

In some embodiments, R is an optionally substituted heterocyclyl. Insome embodiments, R is an optionally substituted 3-7 membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is a substituted 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anunsubstituted 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted heterocyclyl. Insome embodiments, R is an optionally substituted 6 membered saturated orpartially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is an optionally substituted 6 membered partiallyunsaturated heterocyclic ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted 6 membered partially unsaturated heterocyclicring having 2 oxygen atom.

In certain embodiments, R is a 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, R isoxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl,aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl,thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl,thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl,piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl,oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl,tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl,azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl,oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl,dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl,thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl,tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl,oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl,oxathiolanedionyl, piperazinedionyl, morpholinedionyl,thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl,thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, Ris an optionally substituted 5 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, R is an optionally substituted 5-6 memberedpartially unsaturated monocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, R is an optionally substituted tetrahydropyridinyl,dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R is an optionally substituted 8-10 memberedbicyclic saturated or partially unsaturated heterocyclic ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R is an optionally substituted indolinyl. In someembodiments, R is an optionally substituted isoindolinyl. In someembodiments, R is an optionally substituted 1, 2, 3,4-tetrahydroquinoline. In some embodiments, R is an optionallysubstituted 1, 2, 3, 4-tetrahydroisoquinoline.

As defined generally above and herein, each R′ is independently —R,—C(O)R, —CO₂R, or —SO₂R, or:

-   -   two R′ on the same nitrogen are taken together with their        intervening atoms to form an optionally substituted heterocyclic        or heteroaryl ring, or    -   two R′ on the same carbon are taken together with their        intervening atoms to form an optionally substituted aryl,        carbocyclic, heterocyclic, or heteroaryl ring.

In some embodiments, R′ is —R, —C(O)R, —CO₂R, or —SO₂R, wherein R is asdefined above and described herein.

In some embodiments, R′ is —R, wherein R is as defined and describedabove and herein. In some embodiments, R′ is hydrogen.

In some embodiments, R′ is —C(O)R, wherein R is as defined above anddescribed herein. In some embodiments, R′ is —CO₂R, wherein R is asdefined above and described herein. In some embodiments, R′ is —SO₂R,wherein R is as defined above and described herein.

In some embodiments, two R′ on the same nitrogen are taken together withtheir intervening atoms to form an optionally substituted heterocyclicor heteroaryl ring. In some embodiments, two R′ on the same carbon aretaken together with their intervening atoms to form an optionallysubstituted aryl, carbocyclic, heterocyclic, or heteroaryl ring.

As defined generally above and herein, -Cy- is an optionally substitutedbivalent ring selected from phenylene, carbocyclylene, arylene,heteroarylene, or heterocyclylene.

In some embodiments, -Cy- is optionally substituted phenylene. In someembodiments, -Cy- is optionally substituted carbocyclylene. In someembodiments, -Cy- is optionally substituted arylene. In someembodiments, -Cy- is optionally substituted heteroarylene. In someembodiments, -Cy- is optionally substituted heterocyclylene.

As defined generally above and herein, each of X, Y and Z isindependently —O—, —S—, —N(-L-R¹)—, or L, wherein each of L and R¹ isindependently as defined above and described below.

In some embodiments, X is —O—. In some embodiments, X is —S—. In someembodiments, X is —O— or —S—. In some embodiments, an oligonucleotidecomprises at least one internucleotidic linkage of formula I wherein Xis —O—. In some embodiments, an oligonucleotide comprises at least oneinternucleotidic linkage of formula I wherein X is —S—. In someembodiments, an oligonucleotide comprises at least one internucleotidiclinkage of formula I wherein X is —O—, and at least one internucleotidiclinkage of formula I wherein X is —S—. In some embodiments, anoligonucleotide comprises at least one internucleotidic linkage offormula I wherein X is —O—, and at least one internucleotidic linkage offormula I wherein X is —S—, and at least one internucleotidic linkage offormula I wherein L is an optionally substituted, linear or branchedC₁-C₁₀ alkylene, wherein one or more methylene units of L are optionallyand independently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—.

In some embodiments, X is —N(-L-R¹)—. In some embodiments, X is —N(R¹)—.In some embodiments, X is —N(R′)—. In some embodiments, X is —N(R)—. Insome embodiments, X is —NH—.

In some embodiments, X is L. In some embodiments, X is a covalent bond.In some embodiments, X is or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In someembodiments, X is an optionally substituted C₁-C₁₀ alkylene or C₁-C₁₀alkenylene. In some embodiments, X is methylene.

In some embodiments, Y is —O—. In some embodiments, Y is —S—.

In some embodiments, Y is —N(-L-R¹)—. In some embodiments, Y is —N(R¹)—.In some embodiments, Y is —N(R′)—. In some embodiments, Y is —N(R)—. Insome embodiments, Y is —NH—.

In some embodiments, Y is L. In some embodiments, Y is a covalent bond.In some embodiments, Y is or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In someembodiments, Y is an optionally substituted C₁-C₁₀ alkylene or C₁-C₁₀alkenylene. In some embodiments, Y is methylene.

In some embodiments, Z is —O—. In some embodiments, Z is —S—.

In some embodiments, Z is —N(-L-R¹)—. In some embodiments, Z is —N(R¹)—.In some embodiments, Z is —N(R′)—. In some embodiments, Z is —N(R)—. Insome embodiments, Z is —NH—.

In some embodiments, Z is L. In some embodiments, Z is a covalent bond.In some embodiments, Z is or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In someembodiments, Z is an optionally substituted C₁-C₁₀ alkylene or C₁-C₁₀alkenylene. In some embodiments, Z is methylene.

As defined generally above and herein, L is a covalent bond or anoptionally substituted, linear or branched C₁-C₁₀ alkylene, wherein oneor more methylene units of L are optionally and independently replacedby an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—.

In some embodiments, L is a covalent bond. In some embodiments, L is anoptionally substituted, linear or branched C₁-C₁₀ alkylene, wherein oneor more methylene units of L are optionally and independently replacedby an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—.

In some embodiments, L has the structure of -L¹-V—, wherein:

L¹ is an optionally substituted group selected from

C₁-C₆ alkylene, C₁-C₆ alkenylene, carbocyclylene, arylene, C₁-C₆heteroalkylene, heterocyclylene, and heteroarylene;V is selected from —O—, —S—, —NR′—, C(R′)₂, —S—S—, —B—S—S—C—,

or an optionally substituted group selected from C₁-C₆ alkylene,arylene, C₁-C₆ heteroalkylene, heterocyclylene, and heteroarylene;

A is ═O, ═S, ═NR′, or ═C(R′)₂;

each of B and C is independently —O—, —S—, —NR′—, —C(R′)₂—, or anoptionally substituted group selected from C₁-C₆ alkylene,carbocyclylene, arylene, heterocyclylene, or heteroarylene; andeach R′ is independently as defined above and described herein.

In some embodiments, L¹ is

In some embodiments, L¹ is

wherein Ring Cy′ is an optionally substituted arylene, carbocyclylene,heteroarylene, or heterocyclylene. In some embodiments, L¹ is optionallysubstituted

In some embodiments, L¹ is

In some embodiments, L¹ is connected to X. In some embodiments, L¹ is anoptionally substituted group selected from

and the sulfur atom is connect to V. In some embodiments, L¹ is anoptionally substituted group selected from

and the carbon atom is connect to X.

In some embodiments, L has the structure of:

wherein:

E is —O—, —S—, —NR′— or —C(R′)₂—;

is a single or double bond;the two R^(L1) are taken together with the two carbon atoms to whichthey are bound to form an optionally substituted aryl, carbocyclic,heteroaryl or heterocyclic ring; and each R′ is independently as definedabove and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   is a single or double bond; and    the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))—, or ═C(CF₃)—.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   is a single or double bond;    the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring; and each R′ is    independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   is a single or double bond;    the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring; and each R′ is    independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   is a single or double bond;    the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring; and each R′ is    independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   is a single or double bond;    the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring; and each R′ is    independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments L has the structure f:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   R′ is as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   R′ is as defined above and described herein.

In some embodiments, L has the structure of:

wherein the phenyl ring is optionally substituted. In some embodiments,the phenyl ring is not substituted. In some embodiments, the phenyl ringis substituted.

In some embodiments, L has the structure of:

wherein the phenyl ring is optionally substituted. In some embodiments,the phenyl ring is not substituted. In some embodiments, the phenyl ringis substituted.

In some embodiments, L has the structure of:

wherein:

-   is a single or double bond; and-   the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   is a single or double bond; and-   the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring.

As defined generally above and herein, E is —O—, —S—, —NR′— or —C(R′)₂—,wherein each R′ independently as defined above and described herein. Insome embodiments, E is —O—, —S—, or —NR′—. In some embodiments, E is—O—, —S—, or —NH—. In some embodiments, E is —O—. In some embodiments, Eis —S—. In some embodiments, E is —NH—.

As defined generally above and herein, G is —O—, —S—, or —NR′, whereineach R′ independently as defined above and described herein. In someembodiments, G is —O—, —S—, or —NH—. In some embodiments, G is —O—. Insome embodiments, G is —S—. In some embodiments, G is —NH—.

In some embodiments, L is -L³-G-, wherein:

-   L³ is an optionally substituted C₁-C₅ alkylene or alkenylene,    wherein one or more methylene units are optionally and independently    replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —S(O)—,    —S(O)₂—, or

andwherein each of G, R′ and Ring Cy′ is independently as defined above anddescribed herein.

In some embodiments, L is -L³-S—, wherein L³ is as defined above anddescribed herein. In some embodiments, L is -L³-O—, wherein L³ is asdefined above and described herein. In some embodiments, L is-L³-N(R′)—, wherein each of L³ and R′ is independently as defined aboveand described herein. In some embodiments, L is -L³-NH—, wherein each ofL³ and R′ is independently as defined above and described herein.

In some embodiments, L³ is an optionally substituted C₅ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Ring Cy′ is independently as defined above anddescribed herein. In some embodiments, L³ is an optionally substitutedC₅ alkylene. In some embodiments, -L³-G- is

In some embodiments, L³ is an optionally substituted C₄ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Cy′ is independently as defined above and describedherein.

In some embodiments, -L³-G- is

In some embodiments, L³ is an optionally substituted C₃ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Cy′ is independently as defined above and describedherein.

In some embodiments, -L³-G- is

In some embodiments, L is

In some embodiments, L is

In some embodiments, L is

In some embodiments, L³ is an optionally substituted C₂ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Cy′ is independently as defined above and describedherein.

In some embodiments, -L³-G- is

wherein each of G and Cy′ is independently as defined above anddescribed herein. In some embodiments, L is

In some embodiments, L is -L⁴-G-, wherein L⁴ is an optionallysubstituted C₁-C₂ alkylene; and G is as defined above and describedherein. In some embodiments, L is -L⁴-G-, wherein L⁴ is an optionallysubstituted C₁-C₂ alkylene; G is as defined above and described herein;and G is connected to R¹. In some embodiments, L is -L⁴-G-, wherein L⁴is an optionally substituted methylene; G is as defined above anddescribed herein; and G is connected to R¹. In some embodiments, L is-L⁴-G-, wherein L⁴ is methylene; G is as defined above and describedherein; and G is connected to R¹. In some embodiments, L is -L⁴-G-,wherein L⁴ is an optionally substituted —(CH₂)₂—; G is as defined aboveand described herein; and G is connected to R¹. In some embodiments, Lis -L⁴-G-, wherein L⁴ is —(CH₂)₂—; G is as defined above and describedherein; and G is connected to R¹.

In some embodiments, L is

wherein G is as defined above and described herein, and G is connectedto R¹. In some embodiments, L is

wherein G is as defined above and described herein, and G is connectedto R¹. In some embodiments, L is

wherein G is as defined above and described herein, and G is connectedto R¹. In some embodiments, L is

wherein the sulfur atom is connected to R¹. In some embodiments, L is

wherein the oxygen atom is connected to R¹.

In some embodiments, L is

wherein G is as defined above and described herein.

In some embodiments, L is —S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3)is an optionally substituted, linear or branched, C₁-C₉ alkylene,wherein one or more methylene units are optionally and independentlyreplaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene,—C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—,—C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—,—OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—,—C(O)S—, —OC(O)—, or —C(O)O—, wherein each of R′ and -Cy- isindependently as defined above and described herein. In someembodiments, L is —S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3) is anoptionally substituted C₁-C₆ alkylene. In some embodiments, L is—S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3) is an optionallysubstituted C₁-C₆ alkenylene. In some embodiments, L is —S—R^(L3)— or—S—C(O)—R^(L3)—, wherein R^(L3) is an optionally substituted C₁-C₆alkylene wherein one or more methylene units are optionally andindependently replaced by an optionally substituted C₁-C₆ alkenylene,arylene, or heteroarylene. In some embodiments, In some embodiments,R^(L3) is an optionally substituted —S—(C₁-C₆ alkenylene)-, —S—(C₁-C₆alkylene)-, —S—(C₁-C₆ alkylene)-arylene-(C₁-C₆ alkylene)-,—S—CO-arylene-(C₁-C₆ alkylene)-, or —S—CO—(C₁-C₆alkylene)-arylene-(C₁-C₆ alkylene)-.

In some embodiments, L is

In some embodiments, L is

In some embodiments, L is

In some embodiments,

In some embodiments, the sulfur atom in the L embodiments describedabove and herein is connected to X. In some embodiments, the sulfur atomin the L embodiments described above and herein is connected to R¹.

As defined generally above and herein, R¹ is halogen, R, or anoptionally substituted C₁-C₅₀ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or—C(O)O—, wherein each variable is independently as defined above anddescribed herein. In some embodiments, R¹ is halogen, R, or anoptionally substituted C₁-C₁₀ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or—C(O)O—, wherein each variable is independently as defined above anddescribed herein.

In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is halogen.In some embodiments, R¹ is —F. In some embodiments, R′ is —Cl. In someembodiments, R¹ is —Br. In some embodiments, R¹ is —I.

In some embodiments, R¹ is R wherein R is as defined above and describedherein.

In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is anoptionally substituted group selected from C₁-C₅₀ aliphatic, phenyl,carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R¹ is an optionally substituted C₁-C₅₀ aliphatic.In some embodiments, R¹ is an optionally substituted C₁-C₁₀ aliphatic.In some embodiments, R¹ is an optionally substituted C₁-C₆ aliphatic. Insome embodiments, R¹ is an optionally substituted C₁-C₆ alkyl. In someembodiments, R¹ is optionally substituted, linear or branched hexyl. Insome embodiments, R¹ is optionally substituted, linear or branchedpentyl. In some embodiments, R¹ is optionally substituted, linear orbranched butyl. In some embodiments, R¹ is optionally substituted,linear or branched propyl. In some embodiments, R¹ is optionallysubstituted ethyl. In some embodiments, R¹ is optionally substitutedmethyl.

In some embodiments, R¹ is optionally substituted phenyl. In someembodiments, R¹ is substituted phenyl. In some embodiments, R¹ isphenyl.

In some embodiments, R¹ is optionally substituted carbocyclyl. In someembodiments, R¹ is optionally substituted C₃-C₁₀ carbocyclyl. In someembodiments, R¹ is optionally substituted monocyclic carbocyclyl. Insome embodiments, R¹ is optionally substituted cycloheptyl. In someembodiments, R¹ is optionally substituted cyclohexyl. In someembodiments, R¹ is optionally substituted cyclopentyl. In someembodiments, R¹ is optionally substituted cyclobutyl. In someembodiments, R¹ is an optionally substituted cyclopropyl. In someembodiments, R¹ is optionally substituted bicyclic carbocyclyl.

In some embodiments, R¹ is an optionally substituted C₁-C₅₀ polycyclichydrocarbon. In some embodiments, R¹ is an optionally substituted C₁-C₅₀polycyclic hydrocarbon wherein one or more methylene units areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein eachvariable is independently as defined above and described herein. In someembodiments, R¹ is optionally substituted

In some embodiments, R¹ is

In some embodiments, R¹ is optionally substituted

In some embodiments, R¹ is an optionally substituted C₁-C₅₀ aliphaticcomprising one or more optionally substituted polycyclic hydrocarbonmoieties. In some embodiments, R¹ is an optionally substituted C₁-C₅₀aliphatic comprising one or more optionally substituted polycyclichydrocarbon moieties, wherein one or more methylene units are optionallyand independently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable isindependently as defined above and described herein. In someembodiments, R¹ is an optionally substituted C₁-C₅₀ aliphatic comprisingone or more optionally substituted

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is an optionally substituted aryl. In someembodiments, R¹ is an optionally substituted bicyclic aryl ring.

In some embodiments, R¹ is an optionally substituted heteroaryl. In someembodiments, R¹ is an optionally substituted 5-6 membered monocyclicheteroaryl ring having 1-3 heteroatoms independently selected fromnitrogen, sulfur, or oxygen. In some embodiments, R¹ is a substituted5-6 membered monocyclic heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is an unsubstituted 5-6 membered monocyclic heteroarylring having 1-3 heteroatoms independently selected from nitrogen,sulfur, or oxygen.

In some embodiments, R¹ is an optionally substituted 5 memberedmonocyclic heteroaryl ring having 1-3 heteroatoms independently selectedfrom nitrogen, oxygen or sulfur. In some embodiments, R¹ is anoptionally substituted 6 membered monocyclic heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R¹ is an optionally substituted 5-memberedmonocyclic heteroaryl ring having 1 heteroatom selected from nitrogen,oxygen, or sulfur. In some embodiments, R¹ is selected from pyrrolyl,furanyl, or thienyl.

In some embodiments, R¹ is an optionally substituted 5-memberedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, R¹ is an optionallysubstituted 5-membered heteroaryl ring having 1 nitrogen atom, and anadditional heteroatom selected from sulfur or oxygen. Exemplary R¹groups include optionally substituted pyrazolyl, imidazolyl, thiazolyl,isothiazolyl, oxazolyl or isoxazolyl.

In some embodiments, R¹ is a 6-membered heteroaryl ring having 1-3nitrogen atoms. In other embodiments, R¹ is an optionally substituted6-membered heteroaryl ring having 1-2 nitrogen atoms. In someembodiments, R¹ is an optionally substituted 6-membered heteroaryl ringhaving 2 nitrogen atoms. In certain embodiments, R¹ is an optionallysubstituted 6-membered heteroaryl ring having 1 nitrogen. Exemplary R¹groups include optionally substituted pyridinyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazinyl, or tetrazinyl.

In certain embodiments, R¹ is an optionally substituted 8-10 memberedbicyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anoptionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R¹ is an optionally substituted 5,6-fused heteroaryl ringhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In certain embodiments, R¹ is an optionally substituted5,6-fused heteroaryl ring having 1 heteroatom independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anoptionally substituted indolyl. In some embodiments, R¹ is an optionallysubstituted azabicyclo[3.2.1]octanyl. In certain embodiments, R¹ is anoptionally substituted 5,6-fused heteroaryl ring having 2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is an optionally substituted azaindolyl. In someembodiments, R¹ is an optionally substituted benzimidazolyl. In someembodiments, R¹ is an optionally substituted benzothiazolyl. In someembodiments, R¹ is an optionally substituted benzoxazolyl. In someembodiments, R¹ is an optionally substituted indazolyl. In certainembodiments, R¹ is an optionally substituted 5,6-fused heteroaryl ringhaving 3 heteroatoms independently selected from nitrogen, oxygen, orsulfur.

In certain embodiments, R¹ is an optionally substituted 6,6-fusedheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R¹ is an optionallysubstituted 6,6-fused heteroaryl ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R¹ is an optionally substituted 6,6-fused heteroaryl ringhaving 1 heteroatom independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R¹ is an optionally substituted quinolinyl.In some embodiments, R¹ is an optionally substituted isoquinolinyl.According to one aspect, R¹ is an optionally substituted 6,6-fusedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R¹ is a quinazoline ora quinoxaline.

In some embodiments, R¹ is an optionally substituted heterocyclyl. Insome embodiments, R¹ is an optionally substituted 3-7 membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is a substituted 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anunsubstituted 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R¹ is an optionally substituted heterocyclyl. Insome embodiments, R¹ is an optionally substituted 6 membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is an optionally substituted 6 membered partiallyunsaturated heterocyclic ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anoptionally substituted 6 membered partially unsaturated heterocyclicring having 2 oxygen atoms.

In certain embodiments, R¹ is a 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, R¹ isoxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl,aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl,thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl,thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl,piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl,oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl,tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl,azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl,oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl,dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl,thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl,tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl,oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl,oxathiolanedionyl, piperazinedionyl, morpholinedionyl,thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl,thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, R¹is an optionally substituted 5 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, R¹ is an optionally substituted 5-6 memberedpartially unsaturated monocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, R¹ is an optionally substituted tetrahydropyridinyl,dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R¹ is an optionally substituted 8-10 memberedbicyclic saturated or partially unsaturated heterocyclic ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R¹ is an optionally substituted indolinyl. In someembodiments, R¹ is an optionally substituted isoindolinyl. In someembodiments, R¹ is an optionally substituted 1, 2, 3,4-tetrahydroquinoline. In some embodiments, R¹ is an optionallysubstituted 1, 2, 3, 4-tetrahydroisoquinoline.

In some embodiments, R¹ is an optionally substituted C₁-C₁₀ aliphaticwherein one or more methylene units are optionally and independentlyreplaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene,—C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—,—C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—,—OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—,—C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable is independently asdefined above and described herein. In some embodiments, R¹ is anoptionally substituted C₁-C₁₀ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionally -Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —OC(O)—, or —C(O)O—, wherein eachR′ is independently as defined above and described herein. In someembodiments, R′ is an optionally substituted C₁-C₁₀ aliphatic whereinone or more methylene units are optionally and independently replaced byan optionally -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —OC(O)—, or—C(O)O—, wherein each R′ is independently as defined above and describedherein.

In some embodiments, R¹ is

In some embodiments, R¹ is CH₃—,

In some embodiments, R¹ comprises a terminal optionally substituted—(CH₂)₂— moiety which is connected to L. Exemplary such R¹ groups aredepicted below:

In some embodiments, R¹ comprises a terminal optionally substituted—(CH₂)— moiety which is connected to L. Exemplary such R¹ groups aredepicted below:

In some embodiments, R¹ is —S—R^(L2), wherein R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, and each of R′ and-Cy- is independently as defined above and described herein. In someembodiments, R¹ is —S—R^(L2), wherein the sulfur atom is connected withthe sulfur atom in L group.

In some embodiments, R¹ is —C(O)—R^(L2), wherein R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, and each of R′ and-Cy- is independently as defined above and described herein. In someembodiments, R¹ is —C(O)—R^(L2), wherein the carbonyl group is connectedwith G in L group. In some embodiments, R¹ is —C(O)—R^(L2), wherein thecarbonyl group is connected with the sulfur atom in L group.

In some embodiments, R^(L2) is optionally substituted C₁-C₉ aliphatic.In some embodiments, R^(L2) is optionally substituted C₁-C₉ alkyl. Insome embodiments, R^(L2) is optionally substituted C₁-C₉ alkenyl. Insome embodiments, R^(L2) is optionally substituted C₁-C₉ alkynyl. Insome embodiments, R^(L2) is an optionally substituted C₁-C₉ aliphaticwherein one or more methylene units are optionally and independentlyreplaced by -Cy- or —C(O)—. In some embodiments, R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by -Cy-. In some embodiments,R^(L2) is an optionally substituted C₁-C₉ aliphatic wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted heterocycylene. In some embodiments, R^(L2) is anoptionally substituted C₁-C₉ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted arylene. In some embodiments, R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by an optionally substitutedheteroarylene. In some embodiments, R^(L2) is an optionally substitutedC₁-C₉ aliphatic wherein one or more methylene units are optionally andindependently replaced by an optionally substituted C₃-C₁₀carbocyclylene. In some embodiments, R^(L2) is an optionally substitutedC₁-C₉ aliphatic wherein two methylene units are optionally andindependently replaced by -Cy- or —C(O)—. In some embodiments, R^(L2) isan optionally substituted C₁-C₉ aliphatic wherein two methylene unitsare optionally and independently replaced by -Cy- or —C(O)—. ExemplaryR^(L2) groups are depicted below:

In some embodiments, R¹ is hydrogen, or an optionally substituted groupselected from

—S—(C₁-C₁₀ aliphatic), C₁-C₁₀ aliphatic, aryl, C₁-C₆ heteroalkyl,heteroaryl and heterocyclyl. In some embodiments, R¹ is

—S—(C₁-C₁₀ aliphatic). In some embodiments, R¹ is

In some embodiments, R¹ is an optionally substituted group selected from—S— (C₁-C₆ aliphatic), C₁-C₁₀ aliphatic, C₁-C₆ heteroaliphatic, aryl,heterocyclyl and heteroaryl.

In some embodiments, R¹ is

In some embodiments, the sulfur atom in the R¹ embodiments describedabove and herein is connected with the sulfur atom, G, E, or —C(O)—moiety in the L embodiments described above and herein. In someembodiments, the —C(O)— moiety in the R¹ embodiments described above andherein is connected with the sulfur atom, G, E, or —C(O)— moiety in theL embodiments described above and herein.

In some embodiments, -L-R¹ is any combination of the L embodiments andR¹ embodiments described above and herein.

In some embodiments, -L-R¹ is -L³-G-R¹ wherein each variable isindependently as defined above and described herein.

In some embodiments, -L-R¹ is -L⁴-G-R¹ wherein each variable isindependently as defined above and described herein.

In some embodiments, -L-R¹ is -L³-G-S—R^(L2), wherein each variable isindependently as defined above and described herein.

In some embodiments, -L-R¹ is -L³-G-C(O)—R^(L2), wherein each variableis independently as defined above and described herein.

In some embodiments, -L-R¹ is

wherein R^(L2) is an optionally substituted C₁-C₉ aliphatic wherein oneor more methylene units are optionally and independently replaced by anoptionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—, and each G is independently as defined above and describedherein.

In some embodiments, -L-R¹ is —R^(L3)—S—S—R^(L2), wherein each variableis independently as defined above and described herein. In someembodiments, -L-R¹ is —R^(L3)—C(O)—S—S—R^(L2), wherein each variable isindependently as defined above and described herein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, L has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments —X-L-R¹ has the structure of:

wherein:

-   the phenyl ring is optionally substituted, and-   each of R¹ and X is independently as defined above and described    herein.

In some embodiments, -L-R¹ is

In some embodiments, -L-R¹ is:

In some embodiments, -L-R¹ is CH₃—,

In some embodiments, -L-R¹ is

In some embodiments, -L-R¹ comprises a terminal optionally substituted—(CH₂)₂— moiety which is connected to X. In some embodiments, -L-R¹comprises a terminal —(CH₂)₂— moiety which is connected to X. Exemplarysuch -L-R¹ moieties are depicted below:

In some embodiments, -L-R¹ comprises a terminal optionally substituted—(CH₂)— moiety which is connected to X. In some embodiments, -L-R¹comprises a terminal —(CH₂)— moiety which is connected to X. Exemplarysuch -L-R¹ moieties are depicted below:

In some embodiments, -L-R¹ is

In some embodiments, -L-R¹ is CH₃—,

and X is —S—.

In some embodiments, -L-R¹ is CH₃—,

X is —S—, W is O, Y is —O—, and Z is —O—.

In some embodiments, R¹ is

or —S—(C₁-C₁₀ aliphatic).

In some embodiments, R¹ is

In some embodiments, X is —O— or —S—, and R¹ is

or —S—(C₁-C₁₀ aliphatic).

In some embodiments, X is —O— or —S—, and R¹ is,

—S—(C₁-C₁₀ aliphatic) or —S—(C₁-C₅₀ aliphatic).

In some embodiments, L is a covalent bond and -L-R¹ is R¹.

In some embodiments, -L-R¹ is not hydrogen.

In some embodiments, —X-L-R¹ is R¹ is

—S—(C₁-C₁₀ aliphatic) or —S—(C₁-C₅₀ aliphatic).

In some embodiments, —X-L-R¹ has the structure of

wherein the

the moiety is optionally substituted. In some embodiments, —X-L-R¹ is

In some embodiments, —X-L-R¹ is

In some embodiments, —X-L-R¹ is

In some embodiments, —X-L-R¹ has the structure of

wherein X′ is O or S, Y′ is —O—, —S— or —NR′—, and the

moiety is optionally substituted. In some embodiments, Y′ is —O—, —S— or—NH—. In some embodiments,

In some embodiments,

In some embodiments,

In some embodiments, —X-L-R¹ has the structure of

wherein X′ is O or S, and the

moiety is optionally substituted. In some embodiments,

In some embodiments, —X-L-R¹ is

wherein the

is optionally substituted. In some embodiments, —X-L-R¹ is

wherein the

is substituted. In some embodiments, —X-L-R¹ is

wherein the

is unsubstituted.

In some embodiments, —X-L-R¹ is R¹—C(O)—S-L^(x)-S—, wherein L^(x) is anoptionally substituted group selected from

In some embodiments, L^(x) is

andIn some embodiments, —X-L-R¹ is (CH₃)₃C—S—S-L^(x)-S—. In someembodiments, —X-L-R¹ is R¹—C(═X′)—Y′—C(R)₂—S-L^(x)-S—. In someembodiments, —X-L-R¹ is R—C(═X′)—Y′—CH₂—S-L^(x)-S—. In some embodiments,—X-L-R¹ is

As will be appreciated by a person skilled in the art, many of the—X-L-R¹ groups described herein are cleavable and can be converted to—X⁻ after administration to a subject. In some embodiments, —X-L-R¹ iscleavable. In some embodiments, —X-L-R¹ is —S-L-R¹, and is converted to—S⁻ after administration to a subject. In some embodiments, theconversion is promoted by an enzyme of a subject. As appreciated by aperson skilled in the art, methods of determining whether the —S-L-R¹group is converted to —S⁻ after administration is widely known andpracticed in the art, including those used for studying drug metabolismand pharmacokinetics.

In some embodiments, the internucleotidic linkage having the structureof formula I is

In some embodiments, the internucleotidic linkage of formula I has thestructure of formula I-a:

wherein each variable is independently as defined above and describedherein.

In some embodiments, the internucleotidic linkage of formula I has thestructure of formula I-b:

wherein each variable is independently as defined above and describedherein.

In some embodiments, the internucleotidic linkage of formula I is anphosphorothioate triester linkage having the structure of formula I-c:

wherein:

-   P* is an asymmetric phosphorus atom and is either Rp or Sp;-   L is a covalent bond or an optionally substituted, linear or    branched C₁-C₁₀ alkylene, wherein one or more methylene units of L    are optionally and independently replaced by an optionally    substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,    —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,    —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or    —C(O)O—;-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆    alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—,    —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,    —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,    —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ on the same nitrogen are taken together with their        intervening atoms to form an optionally substituted heterocyclic        or heteroaryl ring, or    -   two R′ on the same carbon are taken together with their        intervening atoms to form an optionally substituted aryl,        carbocyclic, heterocyclic, or heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, or    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic phenyl carbocyclyl, aryl, heteroaryl,    or heterocyclyl;-   each

independently represents a connection to a nucleoside; and

-   R¹ is not —H when L is a covalent bond.

In some embodiments, the internucleotidic linkage having the structureof formula I is

In some embodiments, the internucleotidic linkage having the structureof formula I-c is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising one or more phosphate diesterlinkages, and one or more modified internucleotide linkages having theformula of I-a, I-b, or I-c.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate triesterlinkage having the structure of formula I-c. In some embodiments, thepresent invention provides a chirally controlled oligonucleotidecomprising at least one phosphate diester internucleotidic linkage andat least two phosphorothioate triester linkages having the structure offormula I-c. In some embodiments, the present invention provides achirally controlled oligonucleotide comprising at least one phosphatediester internucleotidic linkage and at least three phosphorothioatetriester linkages having the structure of formula I-c. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising at least one phosphate diesterinternucleotidic linkage and at least four phosphorothioate triesterlinkages having the structure of formula I-c. In some embodiments, thepresent invention provides a chirally controlled oligonucleotidecomprising at least one phosphate diester internucleotidic linkage andat least five phosphorothioate triester linkages having the structure offormula I-c.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found in any of theAppendixes of the application. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide comprising asequence found in Appendix A. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide comprising a sequencefound in Appendix B. In some embodiments, the present invention providesa chirally controlled oligonucleotide comprising a sequence found inAppendix C. In some embodiments, the present invention provides achirally controlled oligonucleotide having a sequence found in any ofthe Appendixes of the application. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide having asequence found in Appendix A. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having a sequence foundin Appendix B. In some embodiments, the present invention provides achirally controlled oligonucleotide having a sequence found in AppendixC.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106). In some embodiments, the presentinvention provides a chirally controlled oligonucleotide comprising asequence found in GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein thesaid sequence has over 50% identity with GCCTCAGTCTGCTTCGCACC (SEQ IDNO: 106). In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein the said sequence hasover 60% identity with GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106). In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising a sequence found in GCCTCAGTCTGCTTCGCACC (SEQID NO: 106), wherein the said sequence has over 70% identity withGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106). In some embodiments, the presentinvention provides a chirally controlled oligonucleotide comprising asequence found in GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein thesaid sequence has over 80% identity with GCCTCAGTCTGCTTCGCACC (SEQ IDNO: 106). In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein the said sequence hasover 90% identity with GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106). In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising a sequence found in GCCTCAGTCTGCTTCGCACC (SEQID NO: 106), wherein the said sequence has over 95% identity withGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106). In some embodiments, the presentinvention provides a chirally controlled oligonucleotide comprising thesequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106). In some embodiments,the present invention provides a chirally controlled oligonucleotidehaving the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106).

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein at least oneinternucleotidic linkage has a chiral linkage phosphorus. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising a sequence found in GCCTCAGTCTGCTTCGCACC (SEQID NO: 106), wherein at least one internucleotidic linkage has thestructure of formula I. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide comprising a sequencefound in GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein eachinternucleotidic linkage has the structure of formula I. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising a sequence found in GCCTCAGTCTGCTTCGCACC (SEQID NO: 106), wherein at least one internucleotidic linkage has thestructure of formula I-c. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide comprising a sequencefound in GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein eachinternucleotidic linkage has the structure of formula I-c. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising a sequence found in GCCTCAGTCTGCTTCGCACC (SEQID NO: 106), wherein at least one internucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein each internucleotidiclinkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein at least oneinternucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein each internucleotidiclinkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein at least oneinternucleotidic linkage has a chiral linkage phosphorus. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC (SEQ IDNO: 106), wherein at least one internucleotidic linkage has thestructure of formula I. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide comprising the sequenceof GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein each internucleotidiclinkage has the structure of formula I. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide comprising thesequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein at least oneinternucleotidic linkage has the structure of formula I-c. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC (SEQ IDNO: 106), wherein each internucleotidic linkage has the structure offormula I-c. In some embodiments, the present invention provides achirally controlled oligonucleotide comprising the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein at least oneinternucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein each internucleotidiclinkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein at least oneinternucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein each internucleotidiclinkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 106), wherein at least one internucleotidic linkage has achiral linkage phosphorus. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein at least oneinternucleotidic linkage has the structure of formula I. In someembodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:106), wherein each internucleotidic linkage has the structure of formulaI. In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 106), wherein at least one internucleotidic linkage has thestructure of formula I-c. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein each internucleotidiclinkage has the structure of formula I-c. In some embodiments, thepresent invention provides a chirally controlled oligonucleotide havingthe sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein at leastone internucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 106), wherein each internucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 106), wherein at least one internucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 106), wherein each internucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 106), wherein at least one linkage phosphorus is Rp. It isunderstood by a person of ordinary skill in the art that in certainembodiments wherein the chirally controlled oligonucleotide comprises anRNA sequence, each T is independently and optionally replaced with U. Insome embodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:106), wherein each linkage phosphorus is Rp. In some embodiments, thepresent invention provides a chirally controlled oligonucleotide havingthe sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein at leastone linkage phosphorus is Sp. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein each linkage phosphorusis Sp. In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 106), wherein the oligonucleotide is a blockmer. In someembodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:106), wherein the oligonucleotide is a stereoblockmer. In someembodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:106), wherein the oligonucleotide is a P-modification blockmer. In someembodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:106), wherein the oligonucleotide is a linkage blockmer. In someembodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:106), wherein the oligonucleotide is an altmer. In some embodiments, thepresent invention provides a chirally controlled oligonucleotide havingthe sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein theoligonucleotide is a stereoaltmer. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide having thesequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein theoligonucleotide is a P-modification altmer. In some embodiments, thepresent invention provides a chirally controlled oligonucleotide havingthe sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein theoligonucleotide is a linkage altmer. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide having thesequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein theoligonucleotide is a unimer. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein the oligonucleotide is astereounimer. In some embodiments, the present invention provides achirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein the oligonucleotide is aP-modification unimer. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein the oligonucleotide is alinkage unimer. In some embodiments, the present invention provides achirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein the oligonucleotide is agapmer. In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 106), wherein the oligonucleotide is a skipmer.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 106), wherein each cytosine is optionally and independentlyreplaced by 5-methylcytosine. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106), wherein at least one cytosine isoptionally and independently replaced by 5-methylcytosine. In someembodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:106), wherein each cytosine is optionally and independently replaced by5-methylcytosine. Exemplary chirally controlled oligonucleotides havingthe sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 106) are depicted inTable 2, below:

TABLE 2 Exemplary chirally controlled oligonucleotides. SEQ Oligo ID NO:Stereochemistry/Sequence Description 101 106All-(Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] All-R 102 106All-(Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] All-S 103 106(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,5R-9S-5R Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 104 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp,5S-9R-5S Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 105 106(Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Rp,1S-17R-1S Rp, Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 106 106(Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,1R-17S-1R Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 107 106(Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,(R/S)₉R Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 108 106(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,(S/R)₉S Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 109 106(Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp,3S-13R-3S Sp)d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 110 106(Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp,3R-13S-3R Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 111 106(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,18S/R¹⁹ Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 112 106(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,18S/R⁹ Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 113 106(Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,18S/R² Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 114 106(Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp,(RRS)₆-R Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 115 106(Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp,S-(RRS)₆ Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 116 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] RR 122 106All-(Rp)-d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C]All-R 123 106(Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Rp,1S-17R-1SRp, Sp)-d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C]124 106All-(Sp)-d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C]All-S 125 All-(Rp)-d[5mCs1As1Ts1G] All-R 126 All-(Rp)-d[Cs2As2Gs2T]All-R 127 All-(Rp)-d[Cs3As3Gs3T] All-R 128 All-(Sp)-d[Cs4As4Gs4T] All-S129 All-(Sp)-d[Cs5As5Gs5T] All-S 130 All-(Sp)-d[Cs6As6Gs6T] All-S 131106 All-(Rp)-d[Gs7Cs7Cs7Ts7Cs7As7Gs7Ts7Cs7Ts7Gs7 All-RCs7Ts7Ts7Cs7Gs7Cs7As7Cs7C] 132 106All-(Sp)-d[Gs7Cs7Cs7Ts7Cs7As7Gs7Ts7Cs7Ts7Gs7 All-SCs7Ts7Ts7Cs7Gs7Cs7As7Cs7C] 133 106(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,5R-9S-5R Rp)-d[Gs15mCs15mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1Gs15mCs1As15mCs15mC] 134 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp,5S-9R-5S Sp)-d[Gs15mCs15mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1Gs15mCs1As15mCs15mC] 135 108All-(Rp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] All-R 136 108All-(Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] All-S 137 108(Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp)- 1S-9R-1Sd[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 138 108(Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp)- 2S-7R-2Sd[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 139 108(Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp)- 1R-9S-1Rd[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 140 108(Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp)- 2R-7S-2Rd[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 141 108(Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp)- 3S-5R-3Sd[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 142 108(Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp)- 3R-5S-3Rd[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 143 108(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp)- (SSR)₃-SSd[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 144 108(Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp)- (RRS)₃-RRd[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 145 109All-(Rp)-d[5mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1 All-RTs15mCs1Gs15mC] 146 All-(Rp)-d[Gs15mCs1Ts1G] All-R 147All-(Rp)-d[5mCs1As1Gs1T] All-R 148 108All-(Rp)-d[5mCs2As2Gs2Ts25mCs2Ts2Gs25mCs2Ts2Ts25mCs2G] All-R 149 108All-(Rp)-d[5mCs4As4Gs4Ts45mCs4Ts4Gs45mCs4Ts4Ts45mCs4G] All-R 150All-(Rp)-d[TsCs1AsT] All-R 151 All-(Sp)-d[Cs1AsGs1T] All-S 152All-(Sp)-d[Cs1AGs1T] All-S 153 All-(Sp)-d[CAs1GsT] All-S 154All-(Rp)-d[Ts1Cs1As1T] All-R 155 All-(Rp)-d[Ts2Gs2As2C] All-R 156All-(Sp)-d[Gs15mCs1Ts1G] All-S 157 All-(Sp)-d[5mCs1As1Gs1T] All-S 158106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCs1GsCsACsC] 159 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp,5S-9R-5S Sp)-d[Gs1Cs1Cs1Ts1CsAsGsTsCsTsGsCsTsTsCs1GsCs2As2Cs2C] 160 106All-(Rp)- All-R(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE)161 106 All-(Sp)- All-S(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE)162 106(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,5R-9S-5RRp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE)163 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp,5S-9R-5SSp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE)164 106(Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Rp,1S-17R-1S Rp, Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 165 106(Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,1R-17S-1R Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 166 106(Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,(R/S)₉R Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 167 106(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,(S/R)₉S Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 168 106(Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp,3S-13R-3S Sp)(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 169 106(Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp,3R-13S-3R Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 170 106(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,18S/R¹⁹ Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 171 106(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,18S/R⁹ Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 172 106(Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,18S/R² Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 173 106(Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp,(RRS)₆-R Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 174 106(Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp,S-(RRS)₆ Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 175 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs] RR(Gs5mCsAs5mCs5mC)_(MOE) 176 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)(Gs15mCs15mCs1Ts15mCs1)_(MOE)d[As1Gs1Ts15mCs1Ts1Gs15m RRCs1Ts1Ts15mCs1] (Gs15mCs1As15mCs15mC)_(MOE) 177 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)(Gs15mCs15mCs1Ts15mCs1)_(MOE)d[AGT5mCTG5mCTT5mC] RR(Gs25mCs2As25mCs25mC)_(MOE) 178 106(Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp,S-(RRS)₆ Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(F )(F: 2-fluorodeoxyribose) 179 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)d[Gs8Cs8Cs8Ts8Cs8As8Gs8Ts8Cs8Ts8Gs8Cs8Ts8Ts8Cs8Gs8Cs RR8As8Cs8C] 180 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)d[Gs9Cs9Cs9Ts9Cs9As9Gs9Ts9Cs9Ts9Gs9Cs9Ts9Ts9Cs9Gs9Cs RR9As9Cs9C] 181 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)d[Gs10Cs10Cs10Ts10Cs10As10Gs10Ts10Cs10Ts10Gs10Cs10Ts10 RRTs10Cs10Gs10Cs10As10Cs10C] 182 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)d[Gs11Cs11Cs11Ts11Cs11As11Gs11Ts11Cs11Ts11Gs11Cs11Ts11 RRTs11Cs11Gs11Cs11As11Cs11C] 183 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)d[Gs12Cs12Cs12Ts12Cs12As12Gs12Ts12Cs12Ts12Gs12Cs12Ts12 RRTs12Cs12Gs12Cs12As12Cs12C] 184 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)d[Gs13Cs13Cs13Ts13Cs13As13Gs13Ts13Cs13Ts13Gs13Cs13Ts13 RRTs13Cs13Gs13Cs13As13Cs13C] 185 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)d[Gs14Cs14Cs14Ts14Cs14As14Gs14Ts14Cs14Ts14Gs14Cs14Ts14 RRTs14Cs14Gs14Cs14As14Cs14C] 186 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)d[Gs15Cs15Cs15Ts15Cs15As15Gs15Ts15Cs15Ts15Gs15Cs15Ts15 RRTs15Cs15Gs15Cs15As15Cs15C] 187 110(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS-(RRS)₅- Rp)d[GsCsCs1TsCsAs]GsUs2CsUsGsd[CsTs3TsCsGs]CsAs4CsC RR 188106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsACsC] 189 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1CsACs 1C] 190106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S d[Gs8Cs8Cs8Ts8Cs8As8Gs8Ts8Cs8Ts8Gs8Cs8Ts8Ts8Cs8Gs8Cs1A Cs8C]191 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S d[Gs9Cs9Cs9Ts9Cs9As9Gs9Ts9Cs9Ts9Gs9Cs9Ts9Ts9Cs9Gs9Cs1A Cs9C]192 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S d[Gs10Cs10Cs10Ts10Cs10As10Gs10Ts10Cs10Ts10Gs10Cs10Ts10Ts10Cs10Gs10Cs1ACs10C] 193 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S d[Gs11Cs11Cs11Ts11Cs11As11Gs11Ts11Cs11Ts11Gs11Cs11Ts11Ts11Cs11Gs11Cs1ACs11C] 194 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S d[Gs12Cs12Cs12Ts12Cs12As12Gs12Ts12Cs12Ts12Gs12Cs12Ts12Ts12Cs12Gs12Cs1ACs12C] 195 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S d[Gs13Cs13Cs13Ts13Cs13As13Gs13Ts13Cs13Ts13Gs13Cs13Ts13Ts13Cs13Gs13Cs1ACs13C] 196 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S d[Gs14Cs14Cs14Ts14Cs14As14Gs14Ts14Cs14Ts14Gs14Cs14Ts14Ts14Cs14Gs14Cs1ACs14C] 197 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S d[Gs15Cs15Cs15Ts15Cs15As15Gs15Ts15Cs15Ts15Gs15Cs15Ts15Ts15Cs15Gs15Cs1ACs15C] 198 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S GsCsCsUsCsAsGsUsCsUsGsCsUsUsCsGsCsACsC 199 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S Gs1Cs1Cs1Us1Cs1As1Gs1Us1Cs1Us1Gs1Cs1Us1Us1Cs1Gs1CsACs 1C 200111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S Gs8Cs8Cs8Us8Cs8As8Gs8Us8Cs8Us8Gs8Cs8Us8Us8Cs8Gs8Cs1AC s8C 201111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S Gs9Cs9Cs9Us9Cs9As9Gs9Us9Cs9Us9Gs9Cs9Us9Us9Cs9Gs9Cs1AC s9C 202111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S Gs10Cs10Cs10Us10Cs10As10Gs10Us10Cs10Us10Gs10Cs10Us10Us10Cs10Gs10Cs1ACs10C 203 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S Gs11Cs11Cs11Us11Cs11As11Gs11Us11Cs11Us11Gs11Cs11Us11Us11Cs11Gs11Cs1ACs11C 204 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S Gs12Cs12Cs12Us12Cs12As12Gs12Us12Cs12Us12Gs12Cs12Us12Us12Cs12Gs12Cs1ACs12C 205 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S Gs13Cs13Cs13Us13Cs13As13Gs13Us13Cs13Us13Gs13Cs13Us13Us13Cs13Gs13Cs1ACs13C 206 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S Gs14Cs14Cs14Us14Cs14As14Gs14Us14Cs14Us14Gs14Cs14Us14Us14Cs14Gs14Cs1ACs14C 207 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S Gs15Cs15Cs15Us15Cs15As15Gs15Us15Cs15Us15Gs15Cs15Us15Us15Cs15Gs15Cs1ACs15C

In some embodiments, a chirally controlled oligonucleotide is designedsuch that one or more nucleotides comprise a phosphorus modificationprone to “autorelease” under certain conditions. That is, under certainconditions, a particular phosphorus modification is designed such thatit self-cleaves from the oligonucleotide to provide, e.g., a phosphatediester such as those found in naturally occurring DNA and RNA. In someembodiments, such a phosphorus modification has a structure of —O-L-R¹,wherein each of L and R¹ is independently as defined above and describedherein. In some embodiments, an autorelease group comprises a morpholinogroup. In some embodiments, an autorelease group is characterized by theability to deliver an agent to the internucleotidic phosphorus linker,which agent facilitates further modification of the phosphorus atom suchas, e.g., desulfurization. In some embodiments, the agent is water andthe further modification is hydrolysis to form a phosphate diester as isfound in naturally occurring DNA and RNA.

In some embodiments, a chirally controlled oligonucleotide is designedsuch that the resulting pharmaceutical properties are improved throughone or more particular modifications at phosphorus. It is welldocumented in the art that certain oligonucleotides are rapidly degradedby nucleases and exhibit poor cellular uptake through the cytoplasmiccell membrane (Poijarvi-Virta et al., Curr. Med. Chem. (2006), 13(28);3441-65; Wagner et al., Med. Res. Rev. (2000), 20(6):417-51; Peyrotteset al., Mini Rev. Med. Chem. (2004), 4(4):395-408; Gosselin et al.,(1996), 43(1):196-208; Bologna et al., (2002), Antisense & Nucleic AcidDrug Development 12:33-41). For instance, Vives et al., (Nucleic AcidsResearch (1999), 27(20):4071-76) found that tert-butyl SATEpro-oligonucleotides displayed markedly increased cellular penetrationcompared to the parent oligonucleotide.

In some embodiments, a modification at a linkage phosphorus ischaracterized by its ability to be transformed to a phosphate diester,such as those present in naturally occurring DNA and RNA, by one or moreesterases, nucleases, and/or cytochrome P450 enzymes, including but notlimited to, those listed in Table 3, below.

TABLE 3 Exemplary enzymes. Family Gene CYP1 CYP1A1, CYP1A2, CYP1B1 CYP2CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19,CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1 CYP3CYP3A4, CYP3A5, CYP3A7, CYP3A43 CYP4 CYP4A11, CYP4A22, CYP4B1, CYP4F2,CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, CYP4X1, CYP4Z1 CYP5CYP5A1 CYP7 CYP7A1, CYP7B1 CYP8 CYP8A1 (prostacyclin synthase), CYP8B1(bile acid biosynthesis) CYP11 CYP11A1, CYP11B1, CYP11B2 CYP17 CYP17A1CYP19 CYP19A1 CYP20 CYP20A1 CYP21 CYP21A2 CYP24 CYP24A1 CYP26 CYP26A1,CYP26B1, CYP26C1 CYP27 CYP27A1 (bile acid biosynthesis), CYP27B1(vitamin D3 1-alpha hydroxylase, activates vitamin D3), CYP27C1 (unknownfunction) CYP39 CYP39A1 CYP46 CYP46A1 CYP51 CYP51A1 (lanosterol 14-alphademethylase)

In some embodiments, a modification at phosphorus results in aP-modification moiety characterized in that it acts as a pro-drug, e.g.,the P-modification moiety facilitates delivery of an oligonucleotide toa desired location prior to removal. For instance, in some embodiments,a P-modification moiety results from PEGylation at the linkagephosphorus. One of skill in the relevant arts will appreciate thatvarious PEG chain lengths are useful and that the selection of chainlength will be determined in part by the result that is sought to beachieved by PEGylation. For instance, in some embodiments, PEGylation iseffected in order to reduce RES uptake and extend in vivo circulationlifetime of an oligonucleotide.

In some embodiments, a PEGylation reagent for use in accordance with thepresent invention is of a molecular weight of about 300 g/mol to about100,000 g/mol. In some embodiments, a PEGylation reagent is of amolecular weight of about 300 g/mol to about 10,000 g/mol. In someembodiments, a PEGylation reagent is of a molecular weight of about 300g/mol to about 5,000 g/mol. In some embodiments, a PEGylation reagent isof a molecular weight of about 500 g/mol. In some embodiments, aPEGylation reagent of a molecular weight of about 1000 g/mol. In someembodiments, a PEGylation reagent is of a molecular weight of about 3000g/mol. In some embodiments, a PEGylation reagent is of a molecularweight of about 5000 g/mol.

In certain embodiments, a PEGylation reagent is PEG500. In certainembodiments, a PEGylation reagent is PEG1000. In certain embodiments, aPEGylation reagent is PEG3000. In certain embodiments, a PEGylationreagent is PEG5000.

In some embodiments, a P-modification moiety is characterized in that itacts as a PK enhancer, e.g., lipids, PEGylated lipids, etc.

In some embodiments, a P-modification moiety is characterized in that itacts as an agent which promotes cell entry and/or endosomal escape, suchas a membrane-disruptive lipid or peptide.

In some embodiments, a P-modification moiety is characterized in that itacts as a targeting agent. In some embodiments, a P-modification moietyis or comprises a targeting agent. The phrase “targeting agent,” as usedherein, is an entity that is associates with a payload of interest(e.g., with an oligonucleotide or oligonucleotide composition) and alsointeracts with a target site of interest so that the payload of interestis targeted to the target site of interest when associated with thetargeting agent to a materially greater extent than is observed underotherwise comparable conditions when the payload of interest is notassociated with the targeting agent. A targeting agent may be, orcomprise, any of a variety of chemical moieties, including, for example,small molecule moieties, nucleic acids, polypeptides, carbohydrates,etc. Targeting agents are described further by Adarsh et al., “OrganelleSpecific Targeted Drug Delivery—A Review,” International Journal ofResearch in Pharmaceutical and Biomedical Sciences, 2011, p. 895.

Exemplary such targeting agents include, but are not limited to,proteins (e.g. Transferrin), oligopeptides (e.g., cyclic and acylicRGD-containing oligopeptides), antibodies (monoclonal and polyclonalantibodies, e.g. IgG, IgA, IgM, IgD, IgE antibodies),sugars/carbohydrates (e.g., monosaccharides and/or oligosaccharides(mannose, mannose-6-phosphate, galactose, and the like)), vitamins(e.g., folate), or other small biomolecules. In some embodiments, atargeting moiety is a steroid molecule (e.g., bile acids includingcholic acid, deoxycholic acid, dehydrocholic acid; cortisone;digoxigenin; testosterone; cholesterol; cationic steroids such ascortisone having a trimethylaminomethyl hydrazide group attached via adouble bond at the 3-position of the cortisone ring, etc.). In someembodiments, a targeting moiety is a lipophilic molecule (e.g.,alicyclic hydrocarbons, saturated and unsaturated fatty acids, waxes,terpenes, and polyalicyclic hydrocarbons such as adamantine andbuckminsterfullerenes). In some embodiments, a lipophilic molecule is aterpenoid such as vitamin A, retinoic acid, retinal, or dehydroretinal.In some embodiments, a targeting moiety is a peptide.

In some embodiments, a P-modification moiety is a targeting agent offormula —X-L-R¹ wherein each of X, L, and R¹ are as defined in Formula Iabove.

In some embodiments, a P-modification moiety is characterized in that itfacilitates cell specific delivery.

In some embodiments, a P-modification moiety is characterized in that itfalls into one or more of the above-described categories. For instance,in some embodiments, a P-modification moiety acts as a PK enhancer and atargeting ligand. In some embodiments, a P-modification moiety acts as apro-drug and an endosomal escape agent. One of skill in the relevantarts would recognize that numerous other such combinations are possibleand are contemplated by the present invention.

Nucleobases

In some embodiments, a nucleobase present in a provided oligonucleotideis a natural nucleobase or a modified nucleobase derived from a naturalnucleobase. Examples include, but are not limited to, uracil, thymine,adenine, cytosine, and guanine having their respective amino groupsprotected by acyl protecting groups, 2-fluorouracil, 2-fluorocytosine,5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidineanalogs such as pseudoisocytosine and pseudouracil and other modifiednucleobases such as 8-substituted purines, xanthine, or hypoxanthine(the latter two being the natural degradation products). Exemplarymodified nucleobases are disclosed in Chiu and Rana, RNA, 2003, 9,1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7,313.

Compounds represented by the following general formulae are alsocontemplated as modified nucleobases:

wherein R⁸ is an optionally substituted, linear or branched groupselected from aliphatic, aryl, aralkyl, aryloxylalkyl, carbocyclyl,heterocyclyl or heteroaryl group having 1 to 15 carbon atoms, including,by way of example only, a methyl, isopropyl, phenyl, benzyl, orphenoxymethyl group; and each of R⁹ and R¹⁰ is independently anoptionally substituted group selected from linear or branched aliphatic,carbocyclyl, aryl, heterocyclyl and heteroaryl.

Modified nucleobases also include expanded-size nucleobases in which oneor more aryl rings, such as phenyl rings, have been added. Nucleic basereplacements described in the Glen Research catalog(www.glenresearch.com); Krueger A T et al, Acc. Chem. Res., 2007, 40,141-150; Kool, E T, Acc. Chem. Res., 2002, 35, 936-943; Benner S. A., etal., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F. E., et al., Curr.Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin. Chem. Biol.,2006, 10, 622-627, are contemplated as useful for the synthesis of thenucleic acids described herein. Some examples of these expanded-sizenucleobases are shown below:

Herein, modified nucleobases also encompass structures that are notconsidered nucleobases but are other moieties such as, but not limitedto, corrin- or porphyrin-derived rings. Porphyrin-derived basereplacements have been described in Morales-Rojas, H and Kool, E T, Org.Lett., 2002, 4, 4377-4380. Shown below is an example of aporphyrin-derived ring which can be used as a base replacement:

In some embodiments, modified nucleobases are of any one of thefollowing structures, optionally substituted:

In some embodiments, a modified nucleobase is fluorescent. Exemplarysuch fluorescent modified nucleobases include phenanthrene, pyrene,stillbene, isoxanthine, isozanthopterin, terphenyl, terthiophene,benzoterthiophene, coumarin, lumazine, tethered stillbene, benzo-uracil,and naphtho-uracil, as shown below:

In some embodiments, a modified nucleobase is unsubstituted. In someembodiments, a modified nucleobase is substituted. In some embodiments,a modified nucleobase is substituted such that it contains, e.g.,heteroatoms, alkyl groups, or linking moieties connected to fluorescentmoieties, biotin or avidin moieties, or other protein or peptides. Insome embodiments, a modified nucleobase is a “universal base” that isnot a nucleobase in the most classical sense, but that functionssimilarly to a nucleobase. One representative example of such auniversal base is 3-nitropyrrole.

In some embodiments, other nucleosides can also be used in the processdisclosed herein and include nucleosides that incorporate modifiednucleobases, or nucleobases covalently bound to modified sugars. Someexamples of nucleosides that incorporate modified nucleobases include4-acetylcytidine; 5-(carboxyhydroxylmethyl)uridine; 2′-O-methylcytidine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; dihydrouridine;2′-O-methylpseudouridine; beta,D-galactosylqueosine;2′-O-methylguanosine; N⁶-isopentenyladenosine; 1-methyladenosine;1-methylpseudouridine; 1-methylguanosine; 1-methylinosine;2,2-dimethylguanosine; 2-methyladenosine; 2-methylguanosine;N⁷-methylguanosine; 3-methyl-cytidine; 5-methylcytidine;5-hydroxymethylcytidine; 5-formylcytosine; 5-carboxylcytosine;N⁶-methyladenosine; 7-methylguanosine; 5-methylaminoethyluridine;5-methoxyaminomethyl-2-thiouridine; beta,D-mannosylqueosine;5-methoxycarbonylmethyluridine; 5-methoxyuridine;2-methylthio-N⁶-isopentenyladenosine;N-((9-beta,D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine;N-((9-beta,D-ribofuranosylpurine-6-yl)-N-methylcarbamoyl)threonine;uridine-5-oxyacetic acid methylester; uridine-5-oxyacetic acid (v);pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine;2-thiouridine; 4-thiouridine; 5-methyluridine;2′-O-methyl-5-methyluridine; and 2′-O-methyluridine.

In some embodiments, nucleosides include 6′-modified bicyclic nucleosideanalogs that have either (R) or (S)-chirality at the 6′-position andinclude the analogs described in U.S. Pat. No. 7,399,845. In otherembodiments, nucleosides include 5′-modified bicyclic nucleoside analogsthat have either (R) or (S)-chirality at the 5′-position and include theanalogs described in US Patent Application Publication No. 20070287831.

In some embodiments, a nucleobase or modified nucleobase comprises oneor more biomolecule binding moieties such as e.g., antibodies, antibodyfragments, biotin, avidin, streptavidin, receptor ligands, or chelatingmoieties. In other embodiments, a nucleobase or modified nucleobase is5-bromouracil, 5-iodouracil, or 2,6-diaminopurine. In some embodiments,a nucleobase or modified nucleobase is modified by substitution with afluorescent or biomolecule binding moiety. In some embodiments, thesubstituent on a nucleobase or modified nucleobase is a fluorescentmoiety. In some embodiments, the substituent on a nucleobase or modifiednucleobase is biotin or avidin.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200;6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062;6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is hereinincorporated by reference in its entirety.

Sugars

The most common naturally occurring nucleotides are comprised of ribosesugars linked to the nucleobases adenosine (A), cytosine (C), guanine(G), and thymine (T) or uracil (U). Also contemplated are modifiednucleotides wherein a phosphate group or linkage phosphorus in thenucleotides can be linked to various positions of a sugar or modifiedsugar. As non-limiting examples, the phosphate group or linkagephosphorus can be linked to the 2′, 3′, 4′ or 5′ hydroxyl moiety of asugar or modified sugar. Nucleotides that incorporate modifiednucleobases as described herein are also contemplated in this context.In some embodiments, nucleotides or modified nucleotides comprising anunprotected —OH moiety are used in accordance with methods of thepresent invention.

Other modified sugars can also be incorporated within a providedoligonucleotide. In some embodiments, a modified sugar contains one ormore substituents at the 2′ position including one of the following: —F;—CF₃, —CN, —N₃, —NO, —NO₂, —OR′, —SR′, or —N(R′)₂, wherein each R′ isindependently as defined above and described herein; —O—(C₁-C₁₀ alkyl),—S—(C₁-C₁₀ alkyl), —NH—(C₁-C₁₀ alkyl), or —N(C₁-C₁₀ alkyl)₂; —O—(C₂-C₁₀alkenyl), —S—(C₂-C₁₀ alkenyl), —NH—(C₂-C₁₀ alkenyl), or —N(C₂-C₁₀alkenyl)₂; —O—(C₂-C₁₀ alkynyl), —S—(C₂-C₁₀ alkynyl), —NH—(C₂-C₁₀alkynyl), or —N(C₂-C₁₀ alkynyl)₂; or —O—(C₁-C₁₀ alkylene) —O—(C₁-C₁₀alkyl), —O—(C₁-C₁₀ alkylene)-NH—(C₁-C₁₀ alkyl) or —O—(C₁-C₁₀alkylene)-NH(C₁-C₁₀ alkyl)₂, —NH—(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl), or—N(C₁-C₁₀ alkyl)C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl), wherein the alkyl,alkylene, alkenyl and alkynyl may be substituted or unsubstituted.Examples of substituents include, and are not limited to,—O(CH₂)_(n)OCH₃, and —O(CH₂)_(n)NH₂, wherein n is from 1 to about 10,MOE, DMAOE, DMAEOE. Also contemplated herein are modified sugarsdescribed in WO 2001/088198; and Martin et al., Helv. Chim. Acta, 1995,78, 486-504. In some embodiments, a modified sugar comprises one or moregroups selected from a substituted silyl group, an RNA cleaving group, areporter group, a fluorescent label, an intercalator, a group forimproving the pharmacokinetic properties of a nucleic acid, a group forimproving the pharmacodynamic properties of a nucleic acid, or othersubstituents having similar properties. In some embodiments,modifications are made at one or more of the the 2′, 3′, 4′, 5′, or 6′positions of the sugar or modified sugar, including the 3′ position ofthe sugar on the 3′-terminal nucleotide or in the 5′ position of the5′-terminal nucleotide.

In some embodiments, the 2′-OH of a ribose is replaced with asubstituent including one of the following: —H, —F; —CF₃, —CN, —N₃, —NO,—NO₂, —OR′, —SR′, or —N(R′)₂, wherein each R′ is independently asdefined above and described herein; —O—(C₁-C₁₀ alkyl), —S—(C₁-C₁₀alkyl), —NH—(C₁-C₁₀ alkyl), or —N(C₁-C₁₀ alkyl)₂; —O—(C₂-C₁₀ alkenyl),—S—(C₂-C₁₀ alkenyl), —NH—(C₂-C₁₀ alkenyl), or —N(C₂-C₁₀ alkenyl)₂;—O—(C₂-C₁₀ alkynyl), —S—(C₂-C₁₀ alkynyl), —NH—(C₂-C₁₀ alkynyl), or—N(C₂-C₁₀ alkynyl)₂; or —O—(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl),—O—(C₁-C₁₀ alkylene)-NH—(C₁-C₁₀ alkyl) or —O—(C₁-C₁₀ alkylene)-NH(C₁-C₁₀alkyl)₂, —NH—(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl), or —N(C₁-C₁₀alkyl)C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl), wherein the alkyl, alkylene,alkenyl and alkynyl may be substituted or unsubstituted. In someembodiments, the 2′-OH is replaced with —H (deoxyribose). In someembodiments, the 2′-OH is replaced with —F. In some embodiments, the2′-OH is replaced with —OR′. In some embodiments, the 2′-OH is replacedwith —OMe. In some embodiments, the 2′-OH is replaced with —OCH₂CH₂OMe.

Modified sugars also include locked nucleic acids (LNAs). In someembodiments, the locked nucleic acid has the structure indicated below.A locked nucleic acid of the structure below is indicated, wherein Barepresents a nucleobase or modified nucleobase as described herein, andwherein R^(2s) is —OCH₂C4′-.

In some embodiments, a modified sugar is an ENA such as those describedin, e.g., Seth et al., J Am Chem Soc. 2010 Oct. 27; 132(42):14942-14950. In some embodiments, a modified sugar is any of those foundin an XNA (xenonucleic acid), for instance, arabinose, anhydrohexitol,threose, 2′fluoroarabinose, or cyclohexene.

Modified sugars include sugar mimetics such as cyclobutyl or cyclopentylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; and 5,359,044. Some modified sugars that arecontemplated include sugars in which the oxygen atom within the ribosering is replaced by nitrogen, sulfur, selenium, or carbon. In someembodiments, a modified sugar is a modified ribose wherein the oxygenatom within the ribose ring is replaced with nitrogen, and wherein thenitrogen is optionally substituted with an alkyl group (e.g., methyl,ethyl, isopropyl, etc).

Non-limiting examples of modified sugars include glycerol, which formglycerol nucleic acid (GNA) analogues. One example of a GNA analogue isshown below and is described in Zhang, R et al., J. Am. Chem. Soc.,2008, 130, 5846-5847; Zhang L, et al., J. Am. Chem. Soc., 2005, 127,4174-4175 and Tsai C H et al., PNAS, 2007, 14598-14603 (X═O⁻):

Another example of a GNA derived analogue, flexible nucleic acid (FNA)based on the mixed acetal aminal of formyl glycerol, is described inJoyce G F et al., PNAS, 1987, 84, 4398-4402 and Heuberger B D andSwitzer C, J. Am. Chem. Soc., 2008, 130, 412-413, and is shown below:

Additional non-limiting examples of modified sugars includehexopyranosyl (6′ to 4′), pentopyranosyl (4′ to 2′), pentopyranosyl (4′to 3′), or tetrofuranosyl (3′ to 2′) sugars. In some embodiments, ahexopyranosyl (6′ to 4′) sugar is of any one of the following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a pentopyranosyl (4′ to 2′) sugar is of any one ofthe following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a pentopyranosyl (4′ to 3′) sugar is of any one ofthe following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a tetrofuranosyl (3′ to 2′) sugar is of either ofthe following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a modified sugar is of any one of the followingformulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, one or more hydroxyl group in a sugar moiety isoptionally and independently replaced with halogen, R′—N(R′)₂, —OR′, or—SR′, wherein each R′ is independently as defined above and describedherein.

In some embodiments, a sugar mimetic is as illustrated below, whereinX^(s) corresponds to the P-modification group “—XLR¹” described herein,Ba is as defined herein, and X¹ is selected from —S—, —Se—, —CH₂—,—NMe-, —NEt- or —NiPr—.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more(e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more), inclusive,of the sugars in a chirally controlled oligonucleotide composition aremodified. In some embodiments, only purine residues are modified (e.g.,about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%/a, 65%, 70%,75%, 80%, 85%, 90%, 95% or more] of the purine residues are modified).In some embodiments, only pyrimidine residues are modified (e.g., about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or more] of the pyridimine residues are modified). Insome embodiments, both purine and pyrimidine residues are modified.

Modified sugars and sugar mimetics can be prepared by methods known inthe art, including, but not limited to: A. Eschenmoser, Science (1999),284:2118; M. Bohringer et al, Helv. Chim. Acta (1992), 75:1416-1477; M.Egli et al, J. Am. Chem. Soc. (2006), 128(33):10847-56; A. Eschenmoserin Chemical Synthesis: Gnosis to Prognosis, C. Chatgilialoglu and V.Sniekus, Ed., (Kluwer Academic, Netherlands, 1996), p. 293; K.-U.Schoning et al, Science (2000), 290:1347-1351; A. Eschenmoser et al,Helv. Chim. Acta (1992), 75:218; J. Hunziker et al, Helv. Chim. Acta(1993), 76:259; G. Otting et al, Helv. Chim. Acta (1993), 76:2701; K.Groebke et al, Helv. Chim. Acta (1998), 81:375; and A. Eschenmoser,Science (1999), 284:2118. Modifications to the 2′ modifications can befound in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and allreferences therein. Specific modifications to the ribose can be found inthe following references: 2′-fluoro (Kawasaki et. al., J. Med. Chem.,1993, 36, 831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79,1930-1938), “LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310). Insome embodiments, a modified sugar is any of those described in PCTPublication No. WO2012/030683, incorporated herein by reference, anddepicted in the FIGS. 26-30 of the present application.

Oligonucleotides

In some embodiments, the present invention provides oligonucleotides andoligonucleotide compositions that are chirally controlled. For instance,in some embodiments, a provided composition contains predeterminedlevels of one or more individual oligonucleotide types, wherein anoligonucleotide type is defined by: 1) base sequence; 2) pattern ofbackbone linkages; 3) pattern of backbone chiral centers; and 4) patternof backbone P-modifications.

In some embodiments, a provided oligonucleotide is a unimer. In someembodiments, a provided oligonucleotide is a P-modification unimer. Insome embodiments, a provided oligonucleotide is a stereounimer. In someembodiments, a provided oligonucleotide is a stereounimer ofconfiguration Rp. In some embodiments, a provided oligonucleotide is astereounimer of configuration Sp.

In some embodiments, a provided oligonucleotide is an altmer. In someembodiments, a provided oligonucleotide is a P-modification altmer. Insome embodiments, a provided oligonucleotide is a stereoaltmer.

In some embodiments, a provided oligonucleotide is a blockmer. In someembodiments, a provided oligonucleotide is a P-modification blockmer. Insome embodiments, a provided oligonucleotide is a stereoblockmer.

In some embodiments, a provided oligonucleotide is a gapmer.

In some embodiments, a provided oligonucleotide is a skipmer.

In some embodiments, a provided oligonucleotide is a combination of oneor more of unimer, altmer, blockmer, gapmer, and skipmer. For instance,in some embodiments, a provided oligonucleotide is both an altmer and agapmer. In some embodiments, a provided nucleotide is both a gapmer anda skipmer. One of skill in the chemical and synthetic arts willrecognize that numerous other combinations of patterns are available andare limited only by the commercial availability and/or syntheticaccessibility of constituent parts required to synthesize a providedoligonucleotide in accordance with methods of the present invention.

In some embodiments, a provided oligonucleotide comprises one or moreoptionally substituted nucleotides. In some embodiments, a providedoligonucleotide comprises one or more modified nucleotides. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted nucleosides. In some embodiments, a provided oligonucleotidecomprises one or more modified nucleosides. In some embodiments, aprovided oligonucleotide comprises one or more optionally substitutedLNAs.

In some embodiments, a provided oligonucleotide comprises one or moreoptionally substituted nucleobases. In some embodiments, a providedoligonucleotide comprises one or more optionally substituted naturalnucleobases. In some embodiments, a provided oligonucleotide comprisesone or more optionally substituted modified nucleobases. In someembodiments, a provided oligonucleotide comprises one or more5-methylcytidine; 5-hydroxymethylcytidine, 5-formylcytosine, or5-carboxylcytosine. In some embodiments, a provided oligonucleotidecomprises one or more 5-methylcytidine.

In some embodiments, a provided oligonucleotide comprises one or moreoptionally substituted sugars. In some embodiments, a providedoligonucleotide comprises one or more optionally substituted sugarsfound in naturally occurring DNA and RNA. In some embodiments, aprovided oligonucleotide comprises one or more optionally substitutedribose or deoxyribose. In some embodiments, a provided oligonucleotidecomprises one or more optionally substituted ribose or deoxyribose,wherein one or more hydroxyl groups of the ribose or deoxyribose moietyis optionally and independently replaced by halogen, R′, —N(R′)₂, —OR′,or —SR′, wherein each R′ is independently as defined above and describedherein. In some embodiments, a provided oligonucleotide comprises one ormore optionally substituted deoxyribose, wherein the 2′ position of thedeoxyribose is optionally and independently substituted with halogen,R′, —N(R′)₂, —OR′, or —SR′, wherein each R′ is independently as definedabove and described herein. In some embodiments, a providedoligonucleotide comprises one or more optionally substituteddeoxyribose, wherein the 2′ position of the deoxyribose is optionallyand independently substituted with halogen. In some embodiments, aprovided oligonucleotide comprises one or more optionally substituteddeoxyribose, wherein the 2′ position of the deoxyribose is optionallyand independently substituted with one or more —F. halogen. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OR′, wherein each R′ isindependently as defined above and described herein. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OR′, wherein each R′ isindependently an optionally substituted C₁-C₆ aliphatic. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OR′, wherein each R′ isindependently an optionally substituted C₁-C₆ alkyl. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OMe. In some embodiments,a provided oligonucleotide comprises one or more optionally substituteddeoxyribose, wherein the 2′ position of the deoxyribose is optionallyand independently substituted with —O-methoxyethyl.

In some embodiments, a provided oligonucleotide is single-strandedoligonucleotide.

In some embodiments, a provided oligonucleotide is a hybridizedoligonucleotide strand. In certain embodiments, a providedoligonucleotide is a partially hybridized oligonucleotide strand. Incertain embodiments, a provided oligonucleotide is a completelyhybridized oligonucleotide strand. In certain embodiments, a providedoligonucleotide is a double-stranded oligonucleotide. In certainembodiments, a provided oligonucleotide is a triple-strandedoligonucleotide (e.g., a triplex).

In some embodiments, a provided oligonucleotide is chimeric. Forexample, in some embodiments, a provided oligonucleotide is DNA-RNAchimera, DNA-LNA chimera, etc.

In some embodiments, any one of the structures comprising anoligonucleotide depicted in WO2012/030683 can be modified in accordancewith methods of the present invention to provide chirally controlledvariants thereof. For example, in some embodiments the chirallycontrolled variants comprise a stereochemical modification at any one ormore of the linkage phosphorus and/or a P-modification at any one ormore of the linkage phosphorus. For example, in some embodiments, aparticular nucleotide unit of a oligonucleotide of WO2012/030683 ispreselected to be stereochemically modified at the linkage phosphorus ofthat nucleotide unit and/or P-modified at the linkage phosphorus of thatnucleotide unit. In some embodiments, a chirally controlledoligonucleotide is of any one of the structures depicted in FIGS. 26-30.In some embodiments, a chirally controlled oligonucleotide is a variant(e.g., modified version) of any one of the structures depicted in FIGS.26-30. The disclosure of WO2012/030683 is herein incorporated byreference in its entirety.

In some embodiments, a provided oligonucleotide is a therapeutic agent.

In some embodiments, a provided oligonucleotide is an antisenseoligonucleotide.

In some embodiments, a provided oligonucleotide is an antigeneoligonucleotide.

In some embodiments, a provided oligonucleotide is a decoyoligonucleotide.

In some embodiments, a provided oligonucleotide is part of a DNAvaccine.

In some embodiments, a provided oligonucleotide is an immunomodulatoryoligonucleotide, e.g., immunostimulatory oligonucleotide andimmunoinhibitory oligonucleotide.

In some embodiments, a provided oligonucleotide is an adjuvant.

In some embodiments, a provided oligonucleotide is an aptamer.

In some embodiments, a provided oligonucleotide is a ribozyme.

In some embodiments, a provided oligonucleotide is a deoxyribozyme(DNAzymes or DNA enzymes).

In some embodiments, a provided oligonucleotide is an siRNA.

In some embodiments, a provided oligonucleotide is a microRNA, or miRNA.

In some embodiments, a provided oligonucleotide is a ncRNA (non-codingRNAs), including a long non-coding RNA (lncRNA) and a small non-codingRNA, such as piwi-interacting RNA (piRNA).

In some embodiments, a provided oligonucleotide is complementary to astructural RNA, e.g., tRNA.

In some embodiments, a provided oligonucleotide is a nucleic acidanalog, e.g., GNA, LNA, PNA, TNA and Morpholino.

In some embodiments, a provided oligonucleotide is a P-modified prodrug.

In some embodiments, a provided oligonucleotide is a primer. In someembodiments, a primers is for use in polymerase-based chain reactions(i.e., PCR) to amplify nucleic acids. In some embodiments, a primer isfor use in any known variations of PCR, such as reverse transcriptionPCR (RT-PCR) and real-time PCR.

In some embodiments, a provided oligonucleotide is characterized ashaving the ability to modulate RNase H activation. For example, in someembodiments, RNase H activation is modulated by the presence ofstereocontrolled phosphorothioate nucleic acid analogs, with naturalDNA/RNA being more or equally susceptible than the Rp stereoisomer,which in turn is more susceptible than the corresponding Spstereoisomer.

In some embodiments, a provided oligonucleotide is characterized ashaving the ability to indirectly or directly increase or decreaseactivity of a protein or inhibition or promotion of the expression of aprotein. In some embodiments, a provided oligonucleotide ischaracterized in that it is useful in the control of cell proliferation,viral replication, and/or any other cell signaling process.

In some embodiments, a provided oligonucleotide is from about 2 to about200 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 180 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about160 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 140 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about120 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 100 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about90 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 80 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about70 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 60 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about50 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 40 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about30 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 29 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about28 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 27 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about26 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 25 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about24 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 23 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about22 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 21 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about20 nucleotide units in length.

In some embodiments, a provided oligonucleotide is from about 4 to about200 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 180 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about160 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 140 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about120 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 100 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about90 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 80 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about70 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 60 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about50 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 40 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about30 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 29 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about28 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 27 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about26 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 25 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about24 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 23 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about22 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 21 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about20 nucleotide units in length.

In some embodiments, a provided oligonucleotide is from about 5 to about10 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 10 to about 30 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 15 toabout 25 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide units in length.

In some embodiments, the oligonucleotide is at least 2 nucleotide unitsin length. In some embodiments, the oligonucleotide is at least 3nucleotide units in length. In some embodiments, the oligonucleotide isat least 4 nucleotide units in length. In some embodiments, theoligonucleotide is at least 5 nucleotide units in length. In someembodiments, the oligonucleotide is at least 6 nucleotide units inlength. In some embodiments, the oligonucleotide is at least 7nucleotide units in length. In some embodiments, the oligonucleotide isat least 8 nucleotide units in length. In some embodiments, theoligonucleotide is at least 9 nucleotide units in length. In someembodiments, the oligonucleotide is at least 10 nucleotide units inlength. In some embodiments, the oligonucleotide is at least 11nucleotide units in length. In some embodiments, the oligonucleotide isat least 12 nucleotide units in length. In some embodiments, theoligonucleotide is at least 15 nucleotide units in length. In someembodiments, the oligonucleotide is at least 20 nucleotide units inlength. In some embodiments, the oligonucleotide is at least 25nucleotide units in length. In some other embodiments, theoligonucleotide is at least 30 nucleotide units in length. In some otherembodiments, the oligonucleotide is a duplex of complementary strands ofat least 18 nucleotide units in length. In some other embodiments, theoligonucleotide is a duplex of complementary strands of at least 21nucleotide units in length.

In some embodiments, the 5′-end and/or the 3′-end of a providedoligonucleotide is modified. In some embodiments, the 5′-end and/or the3′-end of a provided oligonucleotide is modified with a terminal capmoiety. Exemplary such modifications, including terminal cap moietiesare extensively described herein and in the art, for example but notlimited to those described in US Patent Application Publication US2009/0023675A1.

Species of Oligonucleotides

In certain embodiments, an oligonucleotide of formula I is of any one ofthe structures shown in Table 2, above and those described in theexamples.

In some embodiments, a provided chirally controlled oligonucleotidecomprises the sequence of, or part of the sequence of mipomersen.Mipomersen is based on the following base sequenceGCCT/UCAGT/UCT/UGCT/UT/UCGCACC (SEQ ID NO: 112). In some embodiments,one or more of any of the nucleotide or linkages may be modified inaccordance of the present invention. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide having thesequence ofG*-C*-C*-U*-C*-dA-dG-dT-dC-dT-dG-dmC-dT-dT-dmC-G*-C*-A*-C*-C* (SEQ IDNO: 113) [d=2′-deoxy, *=2′-O-(2-methoxyethyl)] with 3′→5′phosphorothioate linkages. Exemplary modified mipomersen sequences aredescribed throughout the application, including but not limited to thosein Table 4.

In certain embodiments, a provided oligonucleotide is a mipomersenunimer. In certain embodiments, a provided oligonucleotide is amipomersen unimer of configuration Rp. In certain embodiments, aprovided oligonucleotide is a mipomersen unimer of configuration Sp.

Exemplary chirally controlled oligonucleotides comprising the sequenceof, or part of the sequence of mipomersen is depicted in Table 4, below.

TABLE 4 Exemplary Mipomersen related sequences. SEQ De- ID scrip- Oligo NO: Stereochemistry/Sequence tion 101 106All-(Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] All-R 102 106All-(Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] All-S 103 106(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,5R-9S- Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 5R 104 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp,5S-9R- Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 5S 105 106(Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Rp,1S-17R- Rp, Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 1S 106 106(Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,1R-17S- Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 1R 107 106(Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,(R/S)₉R Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 108 106(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,(S/R)₉S Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 109 106(Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp,3S-13R- Sp)d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 3S 110 106(Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp,3R-13S- Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 3R 111 106(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,18S/R¹⁹ Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 112 106(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,18S/R⁹ Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 113 106(Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,18S/R² Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 114 106(Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp,(RRS)₆- Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] R 115 106(Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp,S-(RRS)₆ Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 116 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (RRS)₅- RR 122 106All-(Rp)- All-Rd[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C] 123 106(Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Rp,1S-17R-Rp, Sp)-d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C] 1S124 106All-(Sp)-d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C]All-S 126 All-(Rp)-d[Cs2As2Gs2T] All-R 127 All-(Rp)-d[Cs3As3Gs3T] All-R128 All-(Sp)-d[Cs4As4Gs4T] All-S 129 All-(Sp)-d[Cs5As5Gs5T] All-S 130All-(Sp)-d[Cs6As6Gs6T] All-S 131 106All-(Rp)-d[Gs7Cs7Cs7Ts7Cs7As7Gs7Ts7Cs7Ts7Gs7Cs7Ts7Ts7Cs7Gs7Cs7As7Cs7C]All-R 132 106All-(Sp)-d[Gs7Cs7Cs7Ts7Cs7As7Gs7Ts7Cs7Ts7Gs7Cs7Ts7Ts7Cs7Gs7Cs7As7Cs7C]All-S 133 106(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,5R-9S- Rp)-d[Gs15mCs15mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1 5RGs15mCs1Ts1Ts15mCs1Gs15mCs1As15mCs15mC] 134 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp,5S-9R- Sp)-d[Gs15mCs15mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1 5SGs15mCs1Ts1Ts15mCs1Gs15mCs1As15mCs15mC] 135 108All-(Rp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] All-R 136 108All-(Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] All-S 137 108(Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp)- 1S-9R-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 1S 138 108(Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp)- 2S-7R-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 2S 139 108(Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp)- 1R-9S-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 1R 140 108(Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp)- 2R-7S-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 2R 141 108(Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp)- 3S-5R-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 3S 142 108(Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp)- 3R-5S-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 3R 143 108(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp)- (SSR)₃-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] SS 144 108(Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp)- (RRS)₃-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] RR 145 109 All-(Rp)- All-Rd[5mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1 Gs15mC] 146All-(Rp)-d[Gs15mCs1Ts1G] All-R 147 All-(Rp)-d[5mCs1As1Gs1T] All-R 148108 All-(Rp)-d[5mCs2As2Gs2Ts25mCs2Ts2Gs25mCs2Ts2Ts25mCs2G] All-R 149 108All-(Rp)-d[5mCs4As4Gs4Ts45mCs4Ts4Gs45mCs4Ts4Ts45mCs4G] All-R 151All-(Sp)-d[Cs1AsGs1T] All-S 152 All-(Sp)-d[Cs1AGs1T] All-S 153All-(Sp)-d[CAs1GsT] All-S 157 All-(Sp)-d[5mCs1As1Gs1T] All-S 158 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCs1GsCsACsC] 4S 159 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp,5S-9R- Sp)-d[Gs1Cs1Cs1Ts1CsAsGsTsCsTsGsCsTsTsCs1GsCs2As2Cs2C] 5S 160 106All-(Rp)- All-R (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 161 106 All-(Sp)- All-S(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 162 106(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,5R-9S- Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs] 5R(Gs5mCsAs5mCs5mC)_(MOE) 163 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp,5S-9R- Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs] 5S(Gs5mCsAs5mCs5mC)_(MOE) 164 106(Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Rp,1S-17R- Rp, Sp)- 1S(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 165 106(Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,1R-17S- Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs] 1R(Gs5mCsAs5mCs5mC)_(MOE) 166 106(Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,(R/S)₉R Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 167 106(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,(S/R)₉S Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 168 106(Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp,3S-13R- Sp)(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs] 3S(Gs5mCsAs5mCs5mC)_(MOE) 169 106(Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp,3R-13S- Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs] 3R(Gs5mCsAs5mCs5mC)_(MOE) 170 106(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,18S/R¹⁹ Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 171 106(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,18S/R⁹ Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 172 106(Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,18S/R² Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 173 106(Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp,(RRS)₆-R Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 174 106(Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp,S-(RRS)₆ Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 175 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs] (RRS)₅-(Gs5mCsAs5mCs5mC)_(MOE) RR 176 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)(Gs15mCs15mCs1Ts15mCs1)_(MOE)d[As1Gs1Ts15mCs1Ts1Gs15m (RRS)₅-Cs1Ts1Ts15mCs1] (Gs15mCs1As15mCs15mC)_(MOE) RR 177 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)(Gs15mCs15mCs1Ts15mCs1)_(MOE)d[AGT5mCTG5mCTT5mC] (RRS)₅-(Gs25mCs2As25mCs25mC)_(MOE) RR 178 106(Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp,S-(RRS)₆ Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(F )(F: 2-fluorodeoxyribose) 179 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)d[Gs8Cs8Cs8Ts8Cs8As8Gs8Ts8Cs8Ts8Gs8Cs8Ts8Ts8Cs8Gs8Cs (RRS)₅-8As8Cs8C] RR 180 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)d[Gs9Cs9Cs9Ts9Cs9As9Gs9Ts9Cs9Ts9Gs9Cs9Ts9Ts9Cs9Gs9Cs (RRS)₅-9As9Cs9C] RR 181 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)d[Gs10Cs10Cs10Ts10Cs10As10Gs10Ts10Cs10Ts10Gs10Cs10Ts10 (RRS)₅-Ts10Cs10Gs10Cs10As10Cs10C] RR 182 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)d[Gs11Cs11Cs11Ts11Cs11As11Gs11Ts11Cs11Ts11Gs11Cs11Ts11 (RRS)₅-Ts11Cs11Gs11Cs11As11Cs11C] RR 183 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)d[Gs12Cs12Cs12Ts12Cs12As12Gs12Ts12Cs12Ts12Gs12Cs12Ts12 (RRS)₅-Ts12Cs12Gs12Cs12As12Cs12C] RR 184 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)d[Gs13Cs13Cs13Ts13Cs13As13Gs13Ts13Cs13Ts13Gs13Cs13Ts13 (RRS)₅-Ts13Cs13Gs13Cs13As13Cs13C] RR 185 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)d[Gs14Cs14Cs14Ts14Cs14As14Gs14Ts14Cs14Ts14Gs14Cs14Ts14 (RRS)₅-Ts14Cs14Gs14Cs14As14Cs14C] RR 186 106(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)d[Gs15Cs15Cs15Ts15Cs15As15Gs15Ts15Cs15Ts15Gs15Cs15Ts15 (RRS)₅-Ts15Cs15Gs15Cs15As15Cs15C] RR 187 110(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRS- Rp)d[GsCsCs1TsCsAs]GsUs2CsUsGsd[CsTs3TsCsGs]CsAs4CsC (RRS)₅- RR 188106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsACsC] 4S 189 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1CsACs 4S 1C]190 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- d[Gs8Cs8Cs8Ts8Cs8As8Gs8Ts8Cs8Ts8Gs8Cs8Ts8Ts8Cs8Gs8Cs1A 4S Cs8C]191 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- d[Gs9Cs9Cs9Ts9Cs9As9Gs9Ts9Cs9Ts9Gs9Cs9Ts9Ts9Cs9Gs9Cs1A 4S Cs9C]192 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- d[Gs10Cs10Cs10Ts10Cs10As10Gs10Ts10Cs10Ts10Gs10Cs10Ts10Ts 4S10Cs10Gs10Cs1ACs10C] 193 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- d[Gs11Cs11Cs11Ts11Cs11As11Gs11Ts11Cs11Ts11Gs11Cs11Ts11Ts 4S11Cs11Gs11Cs1ACs11C] 194 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- d[Gs12Cs12Cs12Ts12Cs12As12Gs12Ts12Cs12Ts12Gs12Cs12Ts12Ts 4S12Cs12Gs12Cs1ACs12C] 195 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- d[Gs13Cs13Cs13Ts13Cs13As13Gs13Ts13Cs13Ts13Gs13Cs13Ts13Ts 4S13Cs13Gs13Cs1ACs13C] 196 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- d[Gs14Cs14Cs14Ts14Cs14As14Gs14Ts14Cs14Ts14Gs14Cs14Ts14Ts 4S14Cs14Gs14Cs1ACs14C] 197 106(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- d[Gs15Cs15Cs15Ts15Cs15As15Gs15Ts15Cs15Ts15Gs15Cs15Ts15Ts 4S15Cs15Gs15Cs1ACs15C] 198 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- GsCsCsUsCsAsGsUsCsUsGsCsUsUsCsGsCsACsC 4S 199 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- Gs1Cs1Cs1Us1Cs1As1Gs1Us1Cs1Us1Gs1Cs1Us1Us1Cs1Gs1CsACs 4S 1C 200111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- Gs8Cs8Cs8Us8Cs8As8Gs8Us8Cs8Us8Gs8Cs8Us8Us8Cs8Gs8Cs1AC 4S s8C 201111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- Gs9Cs9Cs9Us9Cs9As9Gs9Us9Cs9Us9Gs9Cs9Us9Us9Cs9Gs9Cs1AC 4S s9C 202111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- Gs10Cs10Cs10Us10Cs10As10Gs10Us10Cs10Us10Gs10Cs10Us10Us 4S10Cs10Gs10Cs1ACs10C 203 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- Gs11Cs11Cs11Us11Cs11As11Gs11Us11Cs11Us11Gs11Cs11Us11Us 4S11Cs11Gs11Cs1ACs11C 204 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- Gs12Cs12Cs12Us12Cs12As12Gs12Us12Cs12Us12Gs12Cs12Us12Us 4S12Cs12Gs12Cs1ACs12C 205 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- Gs13Cs13Cs13Us13Cs13As13Gs13Us13Cs13Us13Gs13Cs13Us13Us 4S13Cs13Gs13Cs1ACs13C 206 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- Gs14Cs14Cs14Us14Cs14As14Gs14Us14Cs14Us14Gs14Cs14Us14Us 4S14Cs14Gs14Cs1ACs14C 207 111(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R- Gs15Cs15Cs15Us15Cs15As15Gs15Us15Cs15Us15Gs15Cs15Us15Us 4S15Cs15Gs15Cs1ACs15C

Oligonucleotide Compositions

The present invention provides compositions comprising or consisting ofa plurality of provided oligonucleotides (e.g., chirally controlledoligonucleotide compositions). In some embodiments, all such providedoligonucleotides are of the same type, i.e., all have the same basesequence, pattern of backbone linkages (i.e., pattern ofinternucleotidic linkage types, for example, phosphate,phosphorothioate, etc), pattern of backbone chiral centers (i.e. patternof linkage phosphorus stereochemistry (Rp/Sp)), and pattern of backbonephosphorus modifications (e.g., pattern of “—XLR¹” groups in formula I).In many embodiments, however, provided compositions comprise a pluralityof oligonucleotides types, typically in pre-determined relative amounts.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a chirally pure mipomersen composition. That is to say,in some embodiments, a provided chirally controlled oligonucleotidecomposition provides mipomersen as a single diastereomer with respect tothe configuration of the linkage phosphorus.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a chirally uniform mipomersen composition. That is tosay, in some embodiments, every linkage phosphorus of mipomersen is inthe Rp configuration or every linkage phosphorus of mipomersen is in theSp configuration.

In some embodiments, a provided chirally controlled oligonucleotidecomposition comprises a combination of one or more providedoligonucleotide types. One of skill in the chemical and medicinal artswill recognize that the selection and amount of each of the one or moretypes of provided oligonucleotides in a provided composition will dependon the intended use of that composition. That is to say, one of skill inthe relevant arts would design a provided chirally controlledoligonucleotide composition such that the amounts and types of providedoligonucleotides contained therein cause the composition as a whole tohave certain desirable characteristics (e.g., biologically desirable,therapeutically desirable, etc.).

In some embodiments, a provided chirally controlled oligonucleotidecomposition comprises a combination of two or more providedoligonucleotide types. In some embodiments, a provided chirallycontrolled oligonucleotide composition comprises a combination of threeor more provided oligonucleotide types. In some embodiments, a providedchirally controlled oligonucleotide composition comprises a combinationof four or more provided oligonucleotide types. In some embodiments, aprovided chirally controlled oligonucleotide composition comprises acombination of five or more provided oligonucleotide types. In someembodiments, a provided chirally controlled oligonucleotide compositioncomprises a combination of six or more provided oligonucleotide types.In some embodiments, a provided chirally controlled oligonucleotidecomposition comprises a combination of seven or more providedoligonucleotide types. In some embodiments, a provided chirallycontrolled oligonucleotide composition comprises a combination of eightor more provided oligonucleotide types. In some embodiments, a providedchirally controlled oligonucleotide composition comprises a combinationof nine or more provided oligonucleotide types. In some embodiments, aprovided chirally controlled oligonucleotide composition comprises acombination of ten or more provided oligonucleotide types. In someembodiments, a provided chirally controlled oligonucleotide compositioncomprises a combination of fifteen or more provided oligonucleotidetypes.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a combination of an amount of chirally uniform mipomersenof the Rp configuration and an amount of chirally uniform mipomersen ofthe Sp configuration.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a combination of an amount of chirally uniform mipomersenof the Rp configuration, an amount of chirally uniform mipomersen of theSp configuration, and an amount of one or more chirally pure mipomersenof a desired diastereomeric form.

Methods for Making Chirally Controlled Oligonucleotides and CompositionsThereof

The present invention provides methods for making chirally controlledoligonucleotides and chirally controlled compositions comprising one ormore specific nucleotide types. As noted above, the phrase“oligonucleotide type,” as used herein, defines an oligonucleotide thathas a particular base sequence, pattern of backbone linkages, pattern ofbackbone chiral centers, and pattern of backbone phosphorusmodifications (e.g., “—XLR¹” groups). Oligonucleotides of a commondesignated “type” are structurally identical to one another with respectto base sequence, pattern of backbone linkages, pattern of backbonechiral centers, and pattern of backbone phosphorus modifications.

In some embodiments, a provided chirally controlled oligonucleotide inthe invention has properties different from those of the correspondingstereorandom oligonucleotide mixture. In some embodiments, a chirallycontrolled oligonucleotide has lipophilicity different from that of thestereorandom oligonucleotide mixture. In some embodiments, a chirallycontrolled oligonucleotide has different retention time on HPLC. In someembodiments, a chirally controlled oligonucleotide may have a peakretention time significantly different from that of the correspondingstereorandom oligonucleotide mixture. During oligonucleotidepurification using HPLC as generally practiced in the art, certainchirally controlled oligonucleotides will be largely if not totallylost. During oligonucleotide purification using HPLC as generallypracticed in the art, certain chirally controlled oligonucleotides willbe largely if not totally lost. One of the consequences is that certaindiastereomers of a stereorandom oligonucleotide mixture (certainchirally controlled oligonucleotides) are not tested in assays. Anotherconsequence is that from batches to batches, due to the inevitableinstrumental and human errors, the supposedly “pure” stereorandomoligonucleotide will have inconsistent compositions in thatdiastereomers in the composition, and their relative and absoluteamounts, are different from batches to batches. The chirally controlledoligonucleotide and chirally controlled oligonucleotide compositionprovided in this invention overcome such problems, as a chirallycontrolled oligonucleotide is synthesized in a chirally controlledfashion as a single diastereomer, and a chirally controlledoligonucleotide composition comprise predetermined levels of one or moreindividual oligonucleotide types.

One of skill in the chemical and synthetic arts will appreciate thatsynthetic methods of the present invention provide for a degree ofcontrol during each step of the synthesis of a provided oligonucleotidesuch that each nucleotide unit of the oligonucleotide can be designedand/or selected in advance to have a particular stereochemistry at thelinkage phosphorus and/or a particular modification at the linkagephosphorus, and/or a particular base, and/or a particular sugar. In someembodiments, a provided oligonucleotide is designed and/or selected inadvance to have a particular combination of stereocenters at the linkagephosphorus of the internucleotidic linkage.

In some embodiments, a provided oligonucleotide made using methods ofthe present invention is designed and/or determined to have a particularcombination of linkage phosphorus modifications. In some embodiments, aprovided oligonucleotide made using methods of the present invention isdesigned and/or determined to have a particular combination of bases. Insome embodiments, a provided oligonucleotide made using methods of thepresent invention is designed and/or determined to have a particularcombination of sugars. In some embodiments, a provided oligonucleotidemade using methods of the present invention is designed and/ordetermined to have a particular combination of one or more of the abovestructural characteristics.

Methods of the present invention exhibit a high degree of chiralcontrol. For instance, methods of the present invention facilitatecontrol of the stereochemical configuration of every single linkagephosphorus within a provided oligonucleotide. In some embodiments,methods of the present invention provide an oligonucleotide comprisingone or more modified internucleotidic linkages independently having thestructure of formula I.

In some embodiments, methods of the present invention provide anoligonucleotide which is a mipomersen unimer. In some embodiments,methods of the present invention provide an oligonucleotide which is amipomersen unimer of configuration Rp. In some embodiments, methods ofthe present invention provide an oligonucleotide which is a mipomersenunimer of configuration Sp.

In some embodiments, methods of the present invention provide a chirallycontrolled oligonucleotide composition, i.e., an oligonucleotidecomposition that contains predetermined levels of individualoligonucleotide types. In some embodiments a chirally controlledoligonucleotide composition comprises one oligonucleotide type. In someembodiments, a chirally controlled oligonucleotide composition comprisesmore than one oligonucleotide type. In some embodiments, a chirallycontrolled oligonucleotide composition comprises a plurality ofoligonucleotide types. Exemplary chirally controlled oligonucleotidecompositions made in accordance with the present invention are describedherein.

In some embodiments, methods of the present invention provide chirallypure mipomersen compositions with respect to the configuration of thelinkage phosphorus. That is to say, in some embodiments, methods of thepresent invention provide compositions of mipomersen wherein mipomersenexists in the composition in the form of a single diastereomer withrespect to the configuration of the linkage phosphorus.

In some embodiments, methods of the present invention provide chirallyuniform mipomersen compositions with respect to the configuration of thelinkage phosphorus. That is to say, in some embodiments, methods of thepresent invention provide compositions of mipomersen in which allnucleotide units therein have the same stereochemistry with respect tothe configuration of the linkage phosphorus, e.g., all nucleotide unitsare of the Rp configuration at the linkage phosphorus or all nucleotideunits are of the Sp configuration at the linkage phosphorus.

In some embodiments, a provided chirally controlled oligonucleotide isover 50% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 55% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 60% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 65% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 70% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 75% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 80% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 85% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 90% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 91% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 92% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 93% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 94% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 95% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 96% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 97% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 98% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 99% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 99.5% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 99.6% pure. In some embodiments, aprovided chirally controlled oligonucleotide is over about 99.7% pure.In some embodiments, a provided chirally controlled oligonucleotide isover about 99.8% pure. In some embodiments, a provided chirallycontrolled oligonucleotide is over about 99.9% pure. In someembodiments, a provided chirally controlled oligonucleotide is over atleast about 99% pure.

In some embodiments, a chirally controlled oligonucleotide compositionis a composition designed to comprise a single oligonucleotide type. Incertain embodiments, such compositions are about 50% diastereomericallypure. In some embodiments, such compositions are about 50%diastereomerically pure. In some embodiments, such compositions areabout 50% diastereomerically pure. In some embodiments, suchcompositions are about 55% diastereomerically pure. In some embodiments,such compositions are about 60% diastereomerically pure. In someembodiments, such compositions are about 65% diastereomerically pure. Insome embodiments, such compositions are about 70% diastereomericallypure. In some embodiments, such compositions are about 75%diastereomerically pure. In some embodiments, such compositions areabout 80% diastereomerically pure. In some embodiments, suchcompositions are about 85% diastereomerically pure. In some embodiments,such compositions are about 90% diastereomerically pure. In someembodiments, such compositions are about 91% diastereomerically pure. Insome embodiments, such compositions are about 92% diastereomericallypure. In some embodiments, such compositions are about 93%diastereomerically pure. In some embodiments, such compositions areabout 94% diastereomerically pure. In some embodiments, suchcompositions are about 95% diastereomerically pure. In some embodiments,such compositions are about 96% diastereomerically pure. In someembodiments, such compositions are about 97% diastereomerically pure. Insome embodiments, such compositions are about 98% diastereomericallypure. In some embodiments, such compositions are about 99%diastereomerically pure. In some embodiments, such compositions areabout 99.5% diastereomerically pure. In some embodiments, suchcompositions are about 99.6% diastereomerically pure. In someembodiments, such compositions are about 99.7% diastereomerically pure.In some embodiments, such compositions are about 99.8%diastereomerically pure. In some embodiments, such compositions areabout 99.9% diastereomerically pure. In some embodiments, suchcompositions are at least about 99% diastereomerically pure.

In some embodiments, a chirally controlled oligonucleotide compositionis a composition designed to comprise multiple oligonucleotide types. Insome embodiments, methods of the present invention allow for thegeneration of a library of chirally controlled oligonucleotides suchthat a pre-selected amount of any one or more chirally controlledoligonucleotide types can be mixed with any one or more other chirallycontrolled oligonucleotide types to create a chirally controlledoligonucleotide composition. In some embodiments, the pre-selectedamount of an oligonucleotide type is a composition having any one of theabove-described diastereomeric purities.

In some embodiments, the present invention provides methods for making achirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved.

When describing the provided methods, the word “cycle” has its ordinarymeaning as understood by a person of ordinary skill in the art. In someembodiments, one round of steps (1)-(4) is referred to as a cycle.

In some embodiments, the present invention provides methods for makingchirally controlled oligonucleotide compositions, comprising steps of:

(a) providing an amount of a first chirally controlled oligonucleotide;and

(b) optionally providing an amount of one or more additional chirallycontrolled oligonucleotides.

In some embodiments, a first chirally controlled oligonucleotide is anoligonucleotide type, as described herein. In some embodiments, a one ormore additional chirally controlled oligonucleotide is a one or moreoligonucleotide type, as described herein.

One of skill in the relevant chemical and synthetic arts will recognizethe degree of versatility and control over structural variation andstereochemical configuration of a provided oligonucleotide whensynthesized using methods of the present invention. For instance, aftera first cycle is complete, a subsequent cycle can be performed using anucleotide unit individually selected for that subsequent cycle which,in some embodiments, comprises a nucleobase and/or a sugar that isdifferent from the first cycle nucleobase and/or sugar. Likewise, thechiral auxiliary used in the coupling step of the subsequent cycle canbe different from the chiral auxiliary used in the first cycle, suchthat the second cycle generates a phosphorus linkage of a differentstereochemical configuration. In some embodiments, the stereochemistryof the linkage phosphorus in the newly formed internucleotidic linkageis controlled by using stereochemically pure phosphoramidites.Additionally, the modification reagent used in the modifying step of asubsequent cycle can be different from the modification reagent used inthe first or former cycle. The cumulative effect of this iterativeassembly approach is such that each component of a providedoligonucleotide can be structurally and configurationally tailored to ahigh degree. An additional advantage to this approach is that the stepof capping minimizes the formation of “n−1” impurities that wouldotherwise make isolation of a provided oligonucleotide extremelychallenging, and especially oligonucleotides of longer lengths.

In some embodiments, an exemplary cycle of the method for makingchirally controlled oligonucleotides is illustrated in Scheme I. InScheme I,

represents the solid support, and optionally a portion of the growingchirally controlled oligonucleotide attached to the solid support. Thechiral auxiliary exemplified has the structure of formula 3-I:

which is further described below. “Cap” is any chemical moietyintroduced to the nitrogen atom by the capping step, and in someembodiments, is an amino protecting group. One of ordinary skill in theart understands that in the first cycle, there may be only onenucleoside attached to the solid support when started, and cycle exitcan be performed optionally before deblocking. As understood by a personof skill in the art, B^(PRO) is a protected base used in oligonucleotidesynthesis. Each step of the above-depicted cycle of Scheme I isdescribed further below.

Synthesis on Solid Support

In some embodiments, the synthesis of a provided oligonucleotide isperformed on solid phase. In some embodiments, reactive groups presenton a solid support are protected. In some embodiments, reactive groupspresent on a solid support are unprotected. During oligonucleotidesynthesis a solid support is treated with various reagents in severalsynthesis cycles to achieve the stepwise elongation of a growingoligonucleotide chain with individual nucleotide units. The nucleosideunit at the end of the chain which is directly linked to the solidsupport is termed “the first nucleoside” as used herein. A firstnucleoside is bound to a solid support via a linker moiety, i.e. adiradical with covalent bonds between either of a CPG, a polymer orother solid support and a nucleoside. The linker stays intact during thesynthesis cycles performed to assemble the oligonucleotide chain and iscleaved after the chain assembly to liberate the oligonucleotide fromthe support.

Solid supports for solid-phase nucleic acid synthesis include thesupports described in, e.g., U.S. Pat. Nos. 4,659,774, 5,141,813,4,458,066; Caruthers U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707,4,668,777, 4,973,679, and 5,132,418; Andrus et al. U.S. Pat. Nos.5,047,524, 5,262,530; and Koster U.S. Pat. No. 4,725,677 (reissued asRe34,069). In some embodiments, a solid phase is an organic polymersupport. In some embodiments, a solid phase is an inorganic polymersupport. In some embodiments, an organic polymer support is polystyrene,aminomethyl polystyrene, a polyethylene glycol-polystyrene graftcopolymer, polyacrylamide, polymethacrylate, polyvinylalcohol, highlycross-linked polymer (HCP), or other synthetic polymers, carbohydratessuch as cellulose and starch or other polymeric carbohydrates, or otherorganic polymers and any copolymers, composite materials or combinationof the above inorganic or organic materials. In some embodiments, aninorganic polymer support is silica, alumina, controlled polyglass(CPG), which is a silica-gel support, or aminopropyl CPG. Other usefulsolid supports include fluorous solid supports (see e.g.,WO/2005/070859), long chain alkylamine (LCAA) controlled pore glass(CPG) solid supports (see e.g., S. P. Adams, K. S. Kavka, E. J. Wykes,S. B. Holder and G. R. Galluppi, J. Am. Chem. Soc., 1983, 105, 661-663;G. R. Gough, M. J. Bruden and P. T. Gilham, Tetrahedron Lett., 1981, 22,4177-4180). Membrane supports and polymeric membranes (see e.g.Innovation and Perspectives in Solid Phase Synthesis, Peptides, Proteinsand Nucleic Acids, ch 21 pp 157-162, 1994, Ed. Roger Epton and U.S. Pat.No. 4,923,901) are also useful for the synthesis of nucleic acids. Onceformed, a membrane can be chemically functionalized for use in nucleicacid synthesis. In addition to the attachment of a functional group tothe membrane, the use of a linker or spacer group attached to themembrane is also used in some embodiments to minimize steric hindrancebetween the membrane and the synthesized chain.

Other suitable solid supports include those generally known in the artto be suitable for use in solid phase methodologies, including, forexample, glass sold as Primer™ 200 support, controlled pore glass (CPG),oxalyl-controlled pore glass (see, e.g., Alul, et al., Nucleic AcidsResearch, 1991, 19, 1527), TentaGel Support—an aminopolyethyleneglycolderivatized support (see, e.g., Wright, et al., Tetrahedron Lett., 1993,34, 3373), and Poros-a copolymer of polystyrene/divinylbenzene.

Surface activated polymers have been demonstrated for use in synthesisof natural and modified nucleic acids and proteins on several solidsupports mediums. A solid support material can be any polymer suitablyuniform in porosity, having sufficient amine content, and sufficientflexibility to undergo any attendant manipulations without losingintegrity. Examples of suitable selected materials include nylon,polypropylene, polyester, polytetrafluoroethylene, polystyrene,polycarbonate, and nitrocellulose. Other materials can serve as a solidsupport, depending on the design of the investigator. In considerationof some designs, for example, a coated metal, in particular gold orplatinum can be selected (see e.g., US publication No. 20010055761). Inone embodiment of oligonucleotide synthesis, for example, a nucleosideis anchored to a solid support which is functionalized with hydroxyl oramino residues. Alternatively, a solid support is derivatized to providean acid labile trialkoxytrityl group, such as a trimethoxytrityl group(TMT). Without being bound by theory, it is expected that the presenceof a trialkoxytrityl protecting group will permit initial detritylationunder conditions commonly used on DNA synthesizers. For a faster releaseof oligonucleotide material in solution with aqueous ammonia, adiglycolate linker is optionally introduced onto the support.

In some embodiments, a provided oligonucleotide alternatively issynthesized from the 5′ to 3′ direction. In some embodiments, a nucleicacid is attached to a solid support through its 5′ end of the growingnucleic acid, thereby presenting its 3′ group for reaction, i.e. using5′-nucleoside phosphoramidites or in enzymatic reaction (e.g. ligationand polymerization using nucleoside 5′-triphosphates). When consideringthe 5′ to 3′ synthesis the iterative steps of the present inventionremain unchanged (i.e. capping and modification on the chiralphosphorus).

Linking Moiety

A linking moiety or linker is optionally used to connect a solid supportto a compound comprising a free nucleophilic moiety. Suitable linkersare known such as short molecules which serve to connect a solid supportto functional groups (e.g., hydroxyl groups) of initial nucleosidesmolecules in solid phase synthetic techniques. In some embodiments, thelinking moiety is a succinamic acid linker, or a succinate linker(—CO—CH₂—CH₂—CO—), or an oxalyl linker (—CO—CO—). In some embodiments,the linking moiety and the nucleoside are bonded together through anester bond. In some embodiments, a linking moiety and a nucleoside arebonded together through an amide bond. In some embodiments, a linkingmoiety connects a nucleoside to another nucleotide or nucleic acid.Suitable linkers are disclosed in, for example, Oligonucleotides AndAnalogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991,Chapter 1 and Solid-Phase Supports for Oligonucleotide Synthesis, Pon,R. T., Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28.

A linker moiety is used to connect a compound comprising a freenucleophilic moiety to another nucleoside, nucleotide, or nucleic acid.In some embodiments, a linking moiety is a phosphodiester linkage. Insome embodiments, a linking moiety is an H-phosphonate moiety. In someembodiments, a linking moiety is a modified phosphorus linkage asdescribed herein. In some embodiments, a universal linker (UnyLinker) isused to attached the oligonucleotide to the solid support (Ravikumar etal., Org. Process Res. Dev., 2008, 12 (3), 399-410). In someembodiments, other universal linkers are used (Pon, R. T., Curr. Prot.Nucleic Acid Chem., 2000, 3.1.1-3.1.28). In some embodiments, variousorthogonal linkers (such as disulfide linkers) are used (Pon, R. T.,Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28).

General Conditions—Solvents for Synthesis

Syntheses of provided oligonucleotides are generally performed inaprotic organic solvents. In some embodiments, a solvent is a nitrilesolvent such as, e.g., acetonitrile. In some embodiments, a solvent is abasic amine solvent such as, e.g., pyridine. In some embodiments, asolvent is an ethereal solvent such as, e.g., tetrahydrofuran. In someembodiments, a solvent is a halogenated hydrocarbon such as, e.g.,dichloromethane. In some embodiments, a mixture of solvents is used. Incertain embodiments a solvent is a mixture of any one or more of theabove-described classes of solvents.

In some embodiments, when an aprotic organic solvent is not basic, abase is present in the reacting step. In some embodiments where a baseis present, the base is an amine base such as, e.g., pyridine,quinoline, or N,N-dimethylaniline. Exemplary other amine bases includepyrrolidine, piperidine, N-methyl pyrrolidine, pyridine, quinoline,N,N-dimethylaminopyridine (DMAP), or N,N-dimethylaniline.

In some embodiments, a base is other than an amine base.

In some embodiments, an aprotic organic solvent is anhydrous. In someembodiments, an anhydrous aprotic organic solvent is freshly distilled.In some embodiments, a freshly distilled anhydrous aprotic organicsolvent is a basic amine solvent such as, e.g., pyridine. In someembodiments, a freshly distilled anhydrous aprotic organic solvent is anethereal solvent such as, e.g., tetrahydrofuran. In some embodiments, afreshly distilled anhydrous aprotic organic solvent is a nitrile solventsuch as, e.g., acetonitrile.

Chiral Reagent

In provided methods, chiral reagents are used to conferstereoselectivity in the production of chirally controlledoligonucleotides. Many different chiral reagents, also referred to bythose of skill in the art and herein as chiral auxiliaries, may be usedin accordance with methods of the present invention. Exemplary suchchiral reagents are described herein and in Wada I, II and III,referenced above. In certain embodiments, a chiral reagent is asdescribed by Wada I. In some embodiments, a chiral reagent for use inaccordance with the methods of the present invention are of Formula 3-I,below:

wherein W¹ and W² are any of —O—, —S—, or —NG⁵-, U₁ and U₃ are carbonatoms which are bonded to U₂ if present, or to each other if r is 0, viaa single, double or triple bond. U₂ is —C—, —CG⁸-, —CG⁸G⁸, —NG⁸-, —N—,—O—, or —S— where r is an integer of 0 to 5 and no more than twoheteroatoms are adjacent. When any one of U₂ is C, a triple bond must beformed between a second instance of U₂, which is C, or to one of U₁ orU₃. Similarly, when any one of U₂ is CG⁸, a double bond is formedbetween a second instance of U₂ which is —CG⁸- or —N—, or to one of U₁or U₃.

In some embodiments, —U₁—(U₂)_(r)—U₃— is —CG³G⁴-CG¹G²-. In someembodiments, —U₁—(U₂)_(r)—U₃— is —CG³=CG¹-. In some embodiments,—U₁—(U₂)_(r)—U₃— is —C≡C—. In some embodiments, —U₁—(U₂)_(r)—U₃— is—CG³=CG⁸-CG¹G²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is—CG³G⁴-O—CG¹G²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is—CG³G⁴-NG⁸-CG¹G²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is—CG³G⁴-N—CG²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is —CG³G⁴-N═CG⁸-CG¹G²-.

As defined herein, G¹, G², G³, G⁴, G⁵, and G⁸ are independentlyhydrogen, or an optionally substituted group selected from alkyl,aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl, andaryl; or two of G¹, G², G³, G⁴, and G⁵ are G⁶ taken together to form anoptionally substituted, saturated, partially unsaturated or unsaturatedcarbocyclic or heteroatom-containing ring of up to about 20 ring atomswhich is monocyclic or polycyclic, and is fused or unfused. In someembodiments, a ring so formed is substituted by oxo, thioxo, alkyl,alkenyl, alkynyl, heteroaryl, or aryl moieties. In some embodiments,when a ring formed by taking two G⁶ together is substituted, it issubstituted by a moiety which is bulky enough to conferstereoselectivity during the reaction.

In some embodiments, a ring formed by taking two of G⁶ together isoptionally substituted cyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl,cyclopentenyl, tetrahydropyranyl, or piperazinyl. In some embodiments, aring formed by taking two of G⁶ together is optionally substitutedcyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl, cyclopentenyl,tetrahydropyranyl, pyrrolidinyl, or piperazinyl.

In some embodiments, G¹ is optionally substituted phenyl. In someembodiments, G¹ is phenyl. In some embodiments, G² is methyl orhydrogen. In some embodiments, G¹ is optionally substituted phenyl andG² is methyl. In some embodiments, G¹ is phenyl and G² is methyl.

In some embodiments, r is 0.

In some embodiments, W¹ is —NG⁵-. In some embodiments, one of G³ and G⁴is taken together with G⁵ to form an optionally substituted pyrrolidinylring. In some embodiments, one of G³ and G⁴ is taken together with G⁵ toform a pyrrolidinyl ring.

In some embodiments, W² is —O—.

In some embodiments, a chiral reagent is a compound of Formula 3-AA:

wherein each variable is independently as defined above and describedherein.

In some embodiments of Formula 3AA, W¹ and W² are independently —NG⁵-,—O—, or —S—; G¹, G², G³, G⁴, and G⁵ are independently hydrogen, or anoptionally substituted group selected from alkyl, aralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, heteroaryl, or aryl; or two of G¹, G²,G³, G⁴, and G⁵ are G⁶ taken together to form an optionally substitutedsaturated, partially unsaturated or unsaturated carbocyclic orheteroatom-containing ring of up to about 20 ring atoms which ismonocyclic or polycyclic, fused or unfused, and no more than four of G¹,G², G³, G⁴, and G⁵ are G⁶. Similarly to the compounds of Formula 3-I,any of G¹, G², G³, G⁴, or G⁵ are optionally substituted by oxo, thioxo,alkyl, alkenyl, alkynyl, heteroaryl, or aryl moieties. In someembodiments, such substitution induces stereoselectivity in chirallycontrolled oligonucleotide production.

In some embodiments, a chiral reagent has one of the following formulae:

In some embodiments, a chiral reagent is an aminoalcohol. In someembodiments, a chiral reagent is an aminothiol. In some embodiments, achiral reagent is an aminophenol. In some embodiments, a chiral reagentis (S)- and (R)-2-methylamino-1-phenylethanol, (1R, 2S)-ephedrine, or(1R, 2S)-2-methylamino-1,2-diphenylethanol.

In some embodiments of the invention, a chiral reagent is a compound ofone of the following formulae:

The choice of chiral reagent, for example, the isomer represented byFormula Q or its stereoisomer, Formula R, permits specific control ofchirality at a linkage phosphorus. Thus, either an Rp or Spconfiguration can be selected in each synthetic cycle, permittingcontrol of the overall three dimensional structure of a chirallycontrolled oligonucleotide. In some embodiments, a chirally controlledoligonucleotide has all Rp stereocenters. In some embodiments of theinvention, a chirally controlled oligonucleotide has all Spstereocenters. In some embodiments of the invention, each linkagephosphorus in the chirally controlled oligonucleotide is independentlyRp or Sp. In some embodiments of the invention, each linkage phosphorusin the chirally controlled oligonucleotide is independently Rp or Sp,and at least one is Rp and at least one is Sp. In some embodiments, theselection of Rp and Sp centers is made to confer a specific threedimensional superstructure to a chirally controlled oligonucleotide.Exemplary such selections are described in further detail herein.

In some embodiments, a chiral reagent for use in accordance with thepresent invention is selected for its ability to be removed at aparticular step in the above-depicted cycle. For example, in someembodiments it is desirable to remove a chiral reagent during the stepof modifying the linkage phosphorus. In some embodiments, it isdesirable to remove a chiral reagent before the step of modifying thelinkage phosphorus. In some embodiments, it is desirable to remove achiral reagent after the step of modifying the linkage phosphorus. Insome embodiments, it is desirable to remove a chiral reagent after afirst coupling step has occurred but before a second coupling step hasoccurred, such that a chiral reagent is not present on the growingoligonucleotide during the second coupling (and likewise for additionalsubsequent coupling steps). In some embodiments, a chiral reagent isremoved during the “deblock” reaction that occurs after modification ofthe linkage phosphorus but before a subsequent cycle begins. Exemplarymethods and reagents for removal are described herein.

In some embodiments, removal of chiral auxiliary is achieved whenperforming the modification and/or deblocking step, as illustrated inScheme I. It can be beneficial to combine chiral auxiliary removaltogether with other transformations, such as modification anddeblocking. A person of ordinary skill in the art would appreciate thatthe saved steps/transformation could improve the overall efficiency ofsynthesis, for instance, with respect to yield and product purity,especially for longer oligonucleotides. One example wherein the chiralauxiliary is removed during modification and/or deblocking isillustrated in Scheme I.

In some embodiments, a chiral reagent for use in accordance with methodsof the present invention is characterized in that it is removable undercertain conditions. For instance, in some embodiments, a chiral reagentis selected for its ability to be removed under acidic conditions. Incertain embodiments, a chiral reagent is selected for its ability to beremoved under mildly acidic conditions. In certain embodiments, a chiralreagent is selected for its ability to be removed by way of an E1elimination reaction (e.g., removal occurs due to the formation of acation intermediate on the chiral reagent under acidic conditions,causing the chiral reagent to cleave from the oligonucleotide). In someembodiments, a chiral reagent is characterized in that it has astructure recognized as being able to accommodate or facilitate an E1elimination reaction. One of skill in the relevant arts will appreciatewhich structures would be envisaged as being prone toward undergoingsuch elimination reactions.

In some embodiments, a chiral reagent is selected for its ability to beremoved with a nucleophile. In some embodiments, a chiral reagent isselected for its ability to be removed with an amine nucleophile. Insome embodiments, a chiral reagent is selected for its ability to beremoved with a nucleophile other than an amine.

In some embodiments, a chiral reagent is selected for its ability to beremoved with a base. In some embodiments, a chiral reagent is selectedfor its ability to be removed with an amine. In some embodiments, achiral reagent is selected for its ability to be removed with a baseother than an amine.

Further Embodiments of Chiral Reagents

In some embodiments, the present invention is directed to a chiralreagent that is used to synthesize chirally controlled oligonucleotides.

In some embodiments, the present invention provides chiral reagents thatare stable to the coupling, capping, modifying and deblocking stepsdescribed above and herein. In some embodiments, the present inventionprovides chiral reagents that are stable to the modifying and deblockingsteps described above and herein. In some embodiments, the presentinvention provides chiral reagents that are stable to the sulfurizationand deblocking steps described above and herein. In some embodiments,the present invention provides chiral reagents that are stable to theoxidation step described above and herein. In some embodiments, such achiral reagent has a structure of formula Z-I.

In some embodiments, the present invention provides chiral reagents thatare removed by treatment with a base and/or a nucleophile. In someembodiments, the present invention provides chiral reagents that areremoved by treatment with a base and/or a nucleophile, and are stable tothe coupling, capping, modifying and deblocking steps described aboveand herein. In some embodiments, the present invention provides chiralreagents that are removed by treatment comprising an amine. In someembodiments, the present invention provides chiral reagents that areremoved by treatment comprising an amine, and are stable to thecoupling, capping, modifying and deblocking steps described above andherein. In some embodiments, the present invention provides chiralreagents that are removed by the deprotection/cleavage conditionsdescribed in this application, and are stable to the coupling, capping,modifying and deblocking steps described above and herein. In someembodiments, such a chiral reagent has a structure of formula Z-I.

In some embodiments, the chiral reagents that are stable to thecoupling, capping, modifying and deblocking steps are used to synthesizechirally controlled oligonucleotides described above and herein. In someembodiments, the chiral reagents that are stable to the coupling,capping, modifying and deblocking steps are used to synthesize chirallycontrolled oligonucleotides described above and herein, wherein thechirally controlled oligonucleotides comprise one or more phosphatediester or phosphorothioate diester linkages. In some embodiments, thechiral reagents that are stable to the coupling, capping, modifying anddeblocking steps are used to synthesize chirally controlledoligonucleotides comprising one or more phosphate diester orphosphorothioate diester linkages, and are not removed until the desiredoligonucleotide lengths have been achieved. In some embodiments, thechiral reagents that are stable to the coupling, capping, modifying anddeblocking steps are used to synthesize chirally controlledoligonucleotides comprising one or more phosphate diester orphosphorothioate diester linkages, and are not removed until after cycleexit. In some embodiments, the chiral reagents that are stable to thecoupling, capping, modifying and deblocking steps are used to synthesizechirally controlled oligonucleotides comprising one or more phosphatediester or phosphorothioate diester linkages, and are not removed untilcleavage from solid support. In some embodiments, the chiral reagentsthat are stable to the coupling, capping, modifying and deblocking stepsare used to synthesize chirally controlled oligonucleotides comprisingone or more phosphate diester or phosphorothioate diester linkages, andare not removed until cleavage from solid support, and the removal isperformed in the same step as cleavage from solid support. In someembodiments, such a chiral reagent has a structure of formula Z-I.

In some embodiments, when a chiral reagent that is stable to thecoupling, capping, modifying and deblocking steps is used inoligonucleotide synthesis, the oligonucleotide with 5′-OH ready forcoupling can be from any synthetic cycle, including those described inSchemes I, I-b, I-c, I-d, Z-1 and Z-2. In some embodiments, theoligonucleotide with 5′-OH for coupling comprises various types ofinternucleotidic linkages as described above and herein. After coupling,the modifying step as described in this application installs the desiredmodification to the linkage phosphorus. The product can either go tocycle exit before/after deblocking, or enter the next cycle afterdeblocking the 5′-OH. It is understood by a person of ordinary skill inthe art that the next cycle can be any of the synthetic cycles describedin this application, including but not limited to those in Schemes I,I-b, I-c, I-d, Z-1 and Z-2.

In some embodiments, a chiral reagent or a salt thereof for use inaccordance with the present invention is of chemical formula (Z-I).

In the formula (Z-I), G^(z1) and G^(z2) are independently a hydrogenatom, a nitro group (—NO₂), a halogen atom, a cyano group (—CN), a groupof formula (Z-II) or (Z-III), or both G¹ and G² taken together to form agroup of formula (Z-IV).

In some embodiments, a group of formula (Z-II) is as depicted below:

wherein G²¹ to G²³ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group.

In some embodiments, a group of formula (Z-III) is as depicted below:

wherein G³¹ to G³³ are independently C₁₋₄ alkyl group, C₆₋₁₄ aryl groupC₁₋₄ alkoxy group, C₇₋₁₄ aralkyl group, C₁₋₄ alkyl C₆₋₁₄ aryl group,C₁₋₄ alkoxy C₆₋₁₄ aryl group, or C₆₋₁₄ aryl C₁₋₄ alkyl group.

In some embodiments, a group of formula (Z-IV) is as depicted below:

wherein G⁴¹ to G⁴⁶ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group.

G^(z3) and G^(z4) are independently a hydrogen atom, C₁₋₃ alkyl group,C₆₋₁₄ aryl group, or both G^(z3) and G^(z4) taken together to form aheteroatom-containing ring that has 3 to 16 carbon atoms, together withthe NH moiety in formula (Z-I).

In some embodiments, a chiral reagent has following chemical formula(Z-I′):

wherein G^(z1) and G^(z2) are same as above. Namely, G^(z1) and G^(z2)are independently a hydrogen atom, a nitro group, a halogen atom, acyano group, a group of formula (Z-II) or (Z-III), or both G^(z1) andG^(z2) taken together to form a group of formula (Z-IV).

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andeach of G^(z1) and G^(z2) is a group of formula (Z-II), wherein G²¹ toG²³ are independently a hydrogen atom, a nitro group, a halogen atom, acyano group or C₁₋₃ alkyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andeach of G^(z1) and G^(z2) is a group of formula (Z-II) and each of G²¹to G²³ is a hydrogen atom.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom, G^(z2) is a group of formula (Z-II), and G²¹to G²³ are independently a hydrogen atom, a nitro group, a halogen atom,a cyano group or C₁₋₃ alkyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom, G^(z2) is a group of formula (Z-II), each ofG²¹ and G²² is a hydrogen atom and G²³ is a nitro group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom and G^(z2) is a group of formula (III), andG³¹ to G³³ are independently C₁₋₄ alkyl group, C₆₋₁₄ aryl group, C₇₋₁₄aralkyl group, C₁₋₄ alkyl C₆₋₁₄ aryl group, C₁₋₄ alkoxy C₆₋₁₄ arylgroup, or C₆₋₁₄ aryl C₁₋₄ alkyl group.

In some embodiments, the chiral reagent has chemical formula (I′) and G¹is a hydrogen atom and G² is a group of formula (III), and G³¹ to G³³are independently C₁₋₄ alkyl group, C₆ aryl group, C₇₋₁₀ aralkyl group,C₁₋₄ alkyl C₆ aryl group, C₁₋₄ alkoxy C₆ aryl group, or C₆ aryl C₁₋₄alkyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom, G^(z2) is a group of formula (Z-III), and G³¹to G³³ are independently C₁₋₄ alkyl group or C₆ aryl group. Examples ofC₁₋₄ alkyl group are methyl group, ethyl group, n-propyl group,iso-propyl group, n-buthyl group and tert-buthyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom, G^(z2) is a group of formula (Z-III), and G³¹to G³³ are independently C₁₋₄ alkyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom, G^(z2) is a group of formula (Z-III), and G³¹and G³³ are C₆ aryl group and G³² is C₁₋₄ alkyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) and G^(z2) are taken together to form a group of formula (Z-IV),and G⁴¹ to G⁴⁶ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₄ alkyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) and G^(z2) are taken together to form a group of formula (Z-IV),wherein each of G⁴¹ to G⁴⁶ is a hydrogen atom.

In certain embodiments, a chiral reagent is selected from one ofchemical formulae 3a, 3b, 5a, Z-5b, 7a, 7b, 9a, 9b, 11a and 11b:

In some embodiments, a nucleoside 3′-phosphoramidite derivative for usein accordance with the present invention is represented by formula(Z-Va) or (Z-Vb):

wherein G^(z1) to G^(z4) are the same as above, G^(z5) is a protectivegroup of the hydroxyl group, and Bs is a group selected from the groupsrepresented by following formula (Z-VI) to (Z-XI), or derivativesthereof.

Examples of Bs are an adenine, a thymine, a cytosine, a guanine, anuracil, a 5-methylcytosine or derivative thereof;R_(z2) is independently hydrogen, —OH, —SH, —NR^(d)R^(d), —N₃, halogen,alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—,heteroaryl-Y¹—, —OR^(b), or —SR^(b), wherein R^(b) is a blocking moiety;Y¹ is O, NR^(d), S, or Se;R^(d) is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,substituted silyl, carbamate, —P(O)(R^(e))₂, or —HP(O)(R^(e));R^(e) is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, or heteroaryl-Y²—, or acation which is Na⁺, Li⁺, or K⁺, or —O⁻;Y² is O, NR^(d), or S;R^(z3) is a group represented by —CH₂—, —(CH₂)₂—, —CH₂NH—, or—CH₂N(CH₃)—.

Examples of G⁵ are trityl, 4-monomethoxytrityl, 4,4′-dimethoxytrityl,4,4′,4″-trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and9-(p-methoxyphenyl)xanthin-9-yl (MOX).

In some embodiments, a nucleoside 3′-phosphoramidite derivative isrepresented by formula (Z-Va′) or (Z-Vb′):

wherein each of G^(z1), G^(z2), G^(z5), Bs, R^(z2), and R^(z3) isindependently as defined above and described herein.

In some embodiments, the invention relates to a method for synthesis ofa chirally controlled oligonucleotide.

In some embodiments, a provided method comprises a first step ofreacting a molecule comprising an achiral H-phosphonate moiety, thefirst activating reagent and a chiral reagent or a salt thereof to forma monomer. In some embodiments, a chiral reagent has chemical formula(Z-I) and the monomer may be represented by formula (Z-Va), (Z-Vb),(Z-Va′), or (Z-Vb′). The monomer reacts with the second activatingreagent and a nucleoside to form a condensed intermediate. In someembodiments, a subsequent step comprises converting the condensedintermediate to the nucleic acid comprising a chiral X-phosphonatemoiety.

In some embodiments, the present methods provide stable and commerciallyavailable materials as starting materials. In some embodiments, thepresent methods provide a stereocontrolled phosphorous atom-modifiedoligonucleotide using an achiral starting material.

As shown in the working examples, in some embodiments methods of thepresent invention do not cause degradation during deprotection steps.Further the method does not require special capping agents to producephosphorus atom-modified oligonucleotide derivatives.

In some embodiments, the present invention provides a method forsynthesis of stereocontrolled phosphorus atom-modified oligonucleotidederivatives using a chiral monomer. In some embodiments, the first stepis reacting a nucleoside 3′-phosphoramidite derivative which isrepresented by formula (Z-Va), (Z-Vb), (Z-Va′), or (Z-Vb′) with thesecond activating reagent and a nucleoside to form a condensedintermediate. The second step is converting the condensed intermediateto the nucleic acid comprising a chiral X-phosphonate moiety.

All publications and patent applications disclosed herein in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

As used in this “Further Embodiments of Chiral Reagents” section, in acondensation reaction, the term “activating reagent” refers to a reagentthat activates a less reactive site and renders it more susceptible toattack by a nucleophile.

As used in this “Further Embodiments of Chiral Reagents” section, an“alkyl” group refers to an aliphatic hydrocarbon group. The alkyl moietymay be a saturated alkyl group (which means that it does not contain anyunits of unsaturation, e.g. carbon-carbon double bonds or carbon-carbontriple bonds) or the alkyl moiety may be an unsaturated alkyl group(which means that it contains at least one unit of unsaturation). Thealkyl moiety, whether saturated or unsaturated, may be branched,straight chain, or include a cyclic portion. The point of attachment ofan alkyl is at a carbon atom that is not part of a ring. The “alkyl”moiety may have 1 to 10 carbon atoms (whenever it appears herein, anumerical range such as “1 to 10” refers to each integer in the givenrange; e.g., “1 to 10 carbon atoms” means that the alkyl group mayconsist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up toand including 10 carbon atoms, although the present definition alsocovers the occurrence of the term “alkyl” where no numerical range isdesignated). Alkyl includes both branched and straight chain alkylgroups. The alkyl group of the compounds described herein may bedesignated as “C₁-C₆ alkyl” or similar designations. By way of exampleonly, “C₁-C₆ alkyl” indicates that there are one, two, three, four,five, or six carbon atoms in the alkyl chain, i.e., the alkyl chain isselected from e.g., methyl, ethyl, propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, and tert-butyl. Typical alkyl groups include, butare in no way limited to, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tertiary butyl, pentyl, hexyl, allyl, cyclopropylmethyl,cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, and the like. Inone aspect, an alkyl is a C₁-C₆ alkyl. C₁₋₃ alkyl group means straightor branched alkyl group that has 1 to 3 carbon atoms. Examples of C₁₋₃alkyl group are methyl, ethyl, propyl and isopropyl. C₁₋₄ alkyl groupmeans straight or branched alkyl group that has 1 to 4 carbon atoms.Examples of C₁₋₄ alkyl group are methyl, ethyl, propyl, isopropyl,butyl, isobutyl, and tert-butyl.

As used in this “Further Embodiments of Chiral Reagents” section, theterm “aryl” refers to an aromatic ring wherein each of the atoms formingthe ring is a carbon atom. Aryl rings are formed by five, six, seven,eight, nine, or more than nine carbon atoms. Aryl groups are asubstituted or unsubstituted. In one aspect, an aryl is a phenyl or anaphthalenyl. Depending on the structure, an aryl group can be amonoradical or a diradical (i.e., an arylene group). In one aspect, anaryl is a C₆-C₁₀ aryl. C₆₋₁₄ aryl group means aryl group that has 6 to14 carbon atoms. The examples of C₆₋₁₄ aryl group are phenyl, biphenyl,naphthyl, anthracyl, indanyl, phthalimidyl, naphthimidyl,phenanthridinyl, and tetrahydronaphthyl.

The term “aralkyl” refers to an alkyl group substituted with an arylgroup. Suitable aralkyl groups include benzyl, picolyl, and the like,all of which may be optionally substituted.

As used in this “Further Embodiments of Chiral Reagents” section, An“acyl moiety” refers to an alkyl(C═O), aryl(C═O), or aralkyl(C═O) group.An acyl moiety can have an intervening moiety (Y) that is oxy, amino,thio, or seleno between the carbonyl and the hydrocarbon group. Forexample, an acyl group can be alkyl-Y—(C═O), aryl-Y—(C═O) oraralkyl-Y—(C═O).

As used in this “Further Embodiments of Chiral Reagents” section,“alkenyl” groups are straight chain, branch chain, and cyclichydrocarbon groups containing at least one carbon-carbon double bond.Alkenyl groups can be substituted.

As used in this “Further Embodiments of Chiral Reagents” section,“alkynyl” groups are straight chain, branch chain, and cyclichydrocarbon groups containing at least one carbon-carbon triple bond.Alkynyl groups can be substituted.

As used in this “Further Embodiments of Chiral Reagents” section, an“alkoxy” group refers to an alklyl group linked to oxygen i.e.(alkyl)-O— group, where alkyl is as defined herein. Examples includemethoxy (—OCH3) or ethoxy (—OCH2CH3) groups.

As used in this “Further Embodiments of Chiral Reagents” section, an“alkenyloxy” group refers to an alkenyl group linked to oxygen i.e.(alkenyl)-O— group, where alkenyl is as defined herein.

As used in this “Further Embodiments of Chiral Reagents” section, an“alkynyloxy” group refers to an alkynyl group linked to oxygen i.e.(alkynyl)-O— group, where alkynyl is as defined herein.

As used in this “Further Embodiments of Chiral Reagents” section, an“aryloxy” group refers to an aryl group linked to oxygen i.e.(aryl)-O-group, where the aryl is as defined herein. An example includesphenoxy (—OC₆H₅) group.

As used in this “Further Embodiments of Chiral Reagents” section, theterm “alkylseleno” refers to an alkyl group having a substituted selenogroup attached thereto i.e. (alkyl)-Se— group, wherein alkyl is definedherein.

As used in this “Further Embodiments of Chiral Reagents” section, theterm “alkenylseleno” refers to an alkenyl group having a substitutedseleno group attached thereto i.e. (alkenyl)-Se— group, wherein alkenylis defined herein.

As used in this “Further Embodiments of Chiral Reagents” section, theterm “alkynylseleno” refers to an alkynyl group having a substitutedseleno group attached thereto i.e. (alkynyl)-Se— group, wherein alkenylis defined herein.

As used in this “Further Embodiments of Chiral Reagents” section, theterm “alkylthio” refers to an alkyl group attached to a bridging sulfuratom i.e. (alkyl)-S-group, wherein alkyl is defined herein. For example,an alkylthio is a methylthio and the like.

As used in this “Further Embodiments of Chiral Reagents” section, theterm “alkenylthio” refers to an alkenyl group attached to a bridgingsulfur atom i.e. (alkenyl)-S— group, wherein alkenyl is defined herein.

As used in this “Further Embodiments of Chiral Reagents” section, theterm “alkynylthio” refers to an alkynyl group attached to a bridgingsulfur atom i.e. (alkynyl)-S— group, wherein alkenyl is defined herein.

As used in this “Further Embodiments of Chiral Reagents” section, theterm “alkylamino” refers to an amino group substituted with at least onealkyl group i.e. —NH(alkyl) or —N(alkyl)2, wherein alkyl is definedherein.

As used in this “Further Embodiments of Chiral Reagents” section, theterm “alkenylamino” refers to an amino group substituted with at leastone alkenyl group Le —NH(alkenyl) or —N(alkenyl)2, wherein alkenyl isdefined herein.

As used in this “Further Embodiments of Chiral Reagents” section, theterm “alkynylamino” refers to an amino group substituted with at leastone alkynyl group i.e. —NH(alkynyl) or —N(alkynyl)2, wherein alkynyl isdefined herein.

As used in this “Further Embodiments of Chiral Reagents” section, theterm “halogen” is intended to include fluorine, chlorine, bromine andiodine.

As used in this “Further Embodiments of Chiral Reagents” section, a“fluorescent group” refers to a molecule that, when excited with lighthaving a selected wavelength, emits light of a different wavelength.Fluorescent groups include, but are not limited to, indole groups,fluorescein, tetramethylrhodamine, Texas Red, BODIPY,5-[(2-aminoethyl)amino]napthalene-1-sulfonic acid (EDANS), coumarin andLucifer yellow.

As used in this “Further Embodiments of Chiral Reagents” section, an“ammonium ion” is a positively charged polyatomic cation of the chemicalformula NH₄ ⁺.

As used in this “Further Embodiments of Chiral Reagents” section, an“alkylammonium ion” is an ammonium ion that has at least one of itshydrogen atoms replaced by an alkyl group, wherein alkyl is definedherein. Examples include triethylammonium ion, N,N-diisopropylethylammonium ion.

As used in this “Further Embodiments of Chiral Reagents” section, an“iminium ion” has the general structure (R^(x))₂C═N(R^(x))₂ ⁺ The R^(x)groups refer to alkyl, alkenyl, alkynyl, aryl groups as defined herein.A “heteroaromatic iminium ion” refers to an imminium ion where thenitrogen and its attached R^(x) groups form a heteroaromatic ring. A“heterocyclic iminium ion” refers to an imminium ion where the nitrogenand its attached R^(x) groups form a heterocyclic ring.

As used in this “Further Embodiments of Chiral Reagents” section, theterms “amino” or “amine” refers to a —N(R^(h))₂ radical group, whereeach R^(h) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,heteroaryl or heteroarylalkyl, unless stated otherwise specifically inthe specification. When a —N(R^(h))₂ group has two R^(h) other thanhydrogen they can be combined with the nitrogen atom to form a 4-, 5-,6-, or 7-membered ring. For example, —N(R^(h))₂ is meant to include, butnot be limited to, 1-pyrrolidinyl and 4-morpholinyl. Any one or more ofthe hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkylare optionally substituted by one or more substituents whichindependently are alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilyl,—OR^(i), —SR^(i), —OC(O)R^(i), —N(R^(i))₂, —C(O)R^(i), —C(O)OR^(i),—OC(O)N(R^(i))₂, —C(O)N(R^(i))₂, —N(R^(i))C(O)OR, —N(R^(i))C(O)R^(i),—N(R^(i))C(O)N(R^(i))₂, N(R^(i))C(NR)N(R^(i))₂, —N(R^(i))S(O)_(t)R^(i)(where t is 1 or 2), —S(O), or —S(O)_(t)N(R^(i))₂ (where t is 1 or 2),where each R^(i) is independently hydrogen, alkyl, fluoroalkyl,carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl or heteroarylalkyl.

As used in this “Further Embodiments of Chiral Reagents” section,“carbamate” as used herein, refers to a moiety attached to an aminogroup which has the formula —C(O)OR where R is alkyl, fluoroalkyl,carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl or heteroarylalkyl. Examples include butare not limited to Boc (tert-butyl-OC(O)—), CBz (benzyl-OC(O)—), Teoc(Me₃SiCH₂CH₂OC(O)—), alloc (allyl-OC(O)—), or Fmoc(9-fluorenylmethyl-OC(O)—) group

As used in this “Further Embodiments of Chiral Reagents” section,“substituted silyl” as used herein, refers to a moiety which has theformula R^(x) ₃Si—. Examples include, but are not limited to, TBDMS(tert-butyldimethylsilyl), TBDPS (tert-butyldiphenylsilyl) or TMS(trimethylsilyl).

As used in this “Further Embodiments of Chiral Reagents” section, theterm “thiol” refers to —SH groups, and include substituted thiol groupsi.e. —SRJ groups, wherein RJ are each independently a substituted orunsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl,heterocyclyl or heterocyclylalkyl group as defined herein.

In some embodiments, the present invention provides a chiral reagent ora salt thereof. In some embodiments, a chiral reagent is of thefollowing chemical formula (Z-I):

wherein G^(z1) and G^(z2) are independently a hydrogen atom, a nitrogroup, a halogen atom, a cyano group (—CN), a group of formula (Z-II) or(Z-III), or both G^(z1) and G^(z2) taken together to form a group offormula (Z-IV). In some embodiments, the term “chiral reagent” is achemical composition which is used to produce stereocontrolledphosphorous atom-modified nucleotide or oligonucleotide derivatives. Achiral reagent reacts with a nucleoside to form a chiral intermediate.

In some embodiments, a group of formula (Z-II) is of the followingformula:

wherein G²¹ to G²³ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group. In some embodiments,examples of G21 to G23 are a hydrogen atom.

In some embodiments, a group of formula (Z-III) is of the followingformula:

wherein G³¹ to G³³ are independently C₁₋₄ alkyl group, C₆₋₁₄ aryl group,C₁₋₄ alkoxy group, C₇₋₁₄ aralkyl group, C₁₋₄ alkyl C₆₋₁₄ aryl group,C₁₋₄ alkoxy C₆₋₁₄ aryl group, or C₆₋₁₄ aryl C₁₋₄ alkyl group. Examplesof C₁₋₄ alkyl C₆₋₁₄ aryl group are methylphenyl group, and ethylphenylgroup. Examples of C₁₋₄ alkoxy C₆₋₁₄ aryl group are methoxyphenyl groupand ethoxyphenyl group. Examples of C₆₋₁₄ aryl C₁₋₄ alkyl groups arebenzyl group and phenylethyl group. In some embodiments, examples of G³¹to G³³ are independently a methyl group and a phenyl group.

In some embodiments, a group of formula (Z-IV) is of the followingformula:

wherein G⁴¹ to G⁴⁶ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group. In some embodiments,examples of G41 to G46 are a hydrogen atom.

G^(z3) and G^(z4) are independently a hydrogen atom, C₁₋₃ alkyl group,C₆₋₁₄ aryl group, or both G^(z3) and G^(z4) taken together to form aheteroatom-containing ring that has 3 to 16 carbon atoms. In someembodiments, examples of G³ and G⁴ are that taken together to form aheteroatom-containing ring that has 3 to 16 carbon atoms with NH moietyin the formula (I).

In certain embodiments, a chiral reagent has following chemical formula(Z-I′).

In the formula (Z-I′), G^(z1) and G^(z2) are same as above and G^(z1)and G^(z2) are independently a hydrogen atom, a nitro group, a halogenatom, a cyano group, a group of formula (Z-II) or (Z-III), or bothG^(z1) and G^(z2) taken together to form a group of formula (Z-IV).

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andeach of G^(z1) and G^(z2) is a group of formula (Z-II), wherein G²¹ toG²³ are independently a hydrogen atom, a nitro group, a halogen atom, acyano group or C₁₋₃ alkyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andeach of G^(z1) and G^(z2) is a group of formula (Z-II) and each of G²¹to G²³ is a hydrogen atom.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom, G^(z2) is a group of formula (Z-II), and G²¹to G²³ are independently a hydrogen atom, a nitro group, a halogen atom,a cyano group or C₁₋₃ alkyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom, G^(z2) is a group of formula (Z-II), each ofG²¹ and G²² is a hydrogen atom and G²³ is a nitro group (—NO₂).

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom and G^(z2) is a group of formula (Z-III), andG³¹ to G³³ are independently C₁₋₄ alkyl group, C₆₋₁₄ aryl group, C₇₋₁₄aralkyl group, C₁₋₄ alkyl C₆₋₁₄ aryl group, C₁₋₄ alkoxy C₆₋₁₄ arylgroup, or C₆₋₁₄ aryl C₁₋₄ alkyl group.

In some embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom, G^(z2) is a group of formula (Z-III), and G³¹to G³³ are independently C₁₋₄ alkyl group or C₆ aryl group (a phenylgroup). Examples of C₁₋₄ alkyl group are methyl group, ethyl group,n-propyl group, iso-propyl group, n-butyl group and tert-butyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom, G^(z2) is a group of formula (Z-III), and G³¹to G³³ are independently C₁₋₂ alkyl group (a methyl group or an ethylgroup) or C₆ aryl group (a phenyl group).

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom, G^(z2) is a group of formula (Z-III), and G³¹to G³³ are independently C₁₋₄ alkyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) is a hydrogen atom, G^(z2) is a group of formula (Z-III), and G³¹and G³³ are C₆ aryl group (a phenyl group) and G³² is C₁₋₂ alkyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) and G^(z2) are taken together to form a group of formula (Z-IV),and G⁴¹ to G⁴⁶ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group.

In certain embodiments, a chiral reagent has chemical formula (Z-I′) andG^(z1) and G^(z2) are taken together to form a group of formula (Z-IV),wherein each of G⁴¹ to G⁴⁶ is a hydrogen atom.

In certain embodiments, a chiral reagent is selected from one ofchemical formulae 3a, 3b, 5a, Z-5b, 7a, 7b, 9a, 9b, 11a and 11b:

Namely, in some embodiments, a chiral reagent is selected from:

-   (S)-2-(Methyldiphenylsilyl)-1-((S)-1-pyrrolidin-2-yl)ethanol (3a),-   (R)-2-(Methyldiphenylsilyl)-1-((R)-1-pyrrolidin-2-yl)ethanol (3b),-   (S)-2-(Trimethylsilyl)-1-((S)-1-pyrrolidin-2-yl)ethanol (5a),-   (R)-2-(Trimethylsilyl)-1-((R)-1-pyrrolidin-2-yl)ethanol (Z-5b),-   (R)-2,2-Diphenyl-1-((S)-pyrrolidin-2-yl)ethanol (7a),-   (S)-2,2-Diphenyl-1-((R)-pyrrolidin-2-yl)ethanol (7b),-   (R)-2-(4-Nitrophenyl)-1-((S)-pyrrolidin-2-yl)ethanol (9a),-   (S)-2-(4-Nitrophenyl)-1-((R)-pyrrolidin-2-yl)ethanol (9b),-   (R)-(9H-Fluororen-9-yl)((S)-pyrrolidin-2-yl)methanol (11a), or-   (S)-(9H-Fluororen-9-yl)((R)-pyrrolidin-2-yl)methanol (11b).

The chiral reagent reacts with a nucleic acid or modified nucleic acidto be an asymmetric auxiliary group. A nucleoside 3′-phosphoramiditederivative, which is an intermediate of manufacturing a stereocontrolledphosphorous atom-modified oligonucleotide derivative, is obtained bychiral reagent reacting with a nucleic acid or modified nucleic acid.

In some embodiments, the invention provides a nucleoside3′-phosphoramidite derivative which is represented by formula (Z-Va) or(Z-Vb). The compounds of formula (Z-Va) and (Z-Vb) are known as monomersthat are used in synthesizing oligonucleotide derivatives. Thesecompounds are also known as oxazaphospholidine monomers. The sugarmoieties of the compounds represented by formula (Z-Vb) are known as BNAand LNA (when R^(z3) is a methylene group).

In the formula (Z-Va) and (Z-Va), G^(z1) to G^(z4) are same as above,G^(z5) is a protective group of the hydroxyl group, and Bs is a groupselected from the groups represented by formula (Z-VI) to (Z-XI) orderivatives thereof.

Examples of Bs are an adenine, a thymine, a cytosine, a guanine, anuracil, a 5-methylcytosine or derivative thereof;R^(z2) is independently hydrogen, —OH, —SH, —NR^(d)R^(d), —N₃, halogen,alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—,heteroaryl-Y¹—, —OR^(b), or —SR^(b), wherein R^(b) is a blocking moiety;Y¹ is O, NR^(d), S, or Se;R^(d) is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,substituted silyl, carbamate, —P(O)(R^(e))₂, or —HP(O)(R^(e));R^(e) is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, or heteroaryl-Y²—, or acation which is Na⁺, Li⁺, or K⁺, or —O⁻;Y² is O, NR^(d), or S;R^(z3) is a group represented by —CH₂—, —(CH₂)₂—, —CH₂NH—, or—CH₂N(CH₃)—.

Examples of G^(z5) is trityl, 4-monomethoxytrityl, 4,4′-dimethoxytrityl,4,4′,4″-trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and9-(p-methoxyphenyl)xanthin-9-yl (MOX).

In some embodiments, Bs is an adenine, a thymine, a cytosine, a guanine,or derivative thereof. In some embodiments, Bs is a nucleobase or amodified nucleobase. Exemplary derivatives are, for instance, thosedisclosed in JP 2005-89441 A, and are represented as follows:

wherein, in the above formula, each of R⁸ to R¹⁰ is independently C₁₋₁₀alkyl, C₆-C₁₀ aryl, C₆-C₁ aralkyl, or C₆-C₁₀ aryloxyalkyl. In someembodiments, R⁸ is methyl, isopropyl, phenyl, benzyl, and phenoxymethyl.In some embodiments, R⁹ and R¹⁰ are C₁₋₄ alkyl group.

In some embodiments, a nucleoside 3′-phosphoramidite derivative isrepresented by formula (Z-Va′) or (Z-Vb′):

wherein, in the formula (Z-Va′) and (Z-Vb′), each of G^(z1), G^(z2),G^(z5), Bs, R^(z2) and R^(z3) are the same as above. In certainembodiments, a nucleoside 3′-phosphoramidite derivative is a chiralmonomer which is used to produce stereocontrolled phosphorousatom-modified nucleotide and oligonucleotide. Examples of the nucleoside3′-phosphoramidite derivatives are represented by the followingformulae: 12a, 12b, 13a, 13b, 14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b,18a, 18b, 19a, 19b, 20a, 20b, 21a, 21b, 22a, 22b, 23a, 23b, 24a, 24b,25a, 25b, 26a, 26b, 27a, 27b, 28a, 28b, 29a, 29b, 30a, 30b, 31a, 31b,32a, 32b, 33a, 33b, 34a, 34b and 35a.

DMTr represents a 4,4′-dimethoxytrityl group and TOM represents atriisopropylsiloxymethyl group.

Examples using a nucleoside 3′-phosphoramidite derivative are disclosedin, e.g, JP 2005-89441 A. By repeating steps of condensation andde-protection, methods of the present invention facilitate lengtheningthe chain of oligonucleotide, as disclosed therein.

In some embodiments, an oligonucleotide is as shown in formula (Z-X):

wherein, in the formula (Z-X), X^(z) represents sulfide (═S), C₁₋₃alkyl, C₁₋₃ alkoxy, C₁₋₃ alkylthio, C₆-C₁₀ aryl, C₆-C₁₀ aralkyl, orC₆-C₁₀ aryloxialkyl. In some embodiments, X^(z) represents sulfide (═S).n^(z) is an integer that represents 1 to 150, 1 to 100, 1 to 50, or 1 to30. In some embodiments, n^(z) is preferably 2 to 100, preferably 10 to100, preferably 10 to 50, and more preferably 15 to 30.

In some embodiments, the present invention provides methods forsynthesis of a stereocontrolled phosphorus atom-modified oligonucleotidederivative. In some embodiments, the first step is a step of reacting amolecule comprising an achiral H-phosphonate moiety, the firstactivating reagent and a chiral reagent or a salt thereof to form amonomer. In some embodiments, the chiral reagent has chemical formula(Z-I) or (Z-I′) and the monomer may be represented by fomura (Z-Va),(Z-Vb), (Z-Va′), or (Z-Vb′). The monomer reacts with the secondactivating reagent and a nucleoside to form a condensed intermediate.Next step is a step of converting the condensed intermediate to thenucleic acid comprising a chiral X-phosphonate moiety. In someembodiments, the methods are as described in WO 2010/064146. In someembodiments, the steps are as described in route A and route B of WO2010/064146.

In some embodiments, the present invention provides a method ofsynthesizing chirally controlled oligonucleotide as illustrated inScheme Z-1 below.

Activation

An achiral H-phosphonate moiety is treated with the first activatingreagent to form the first intermediate. In one embodiment, the firstactivating reagent is added to the reaction mixture during thecondensation step. Use of the first activating reagent is dependent onreaction conditions such as solvents that are used for the reaction.Examples of the first activating reagent are phosgene, trichloromethylchloroformate, bis(trichloromethyl)carbonate (BTC), oxalyl chloride,Ph₃PCl₂, (PhO)₃PCl₂, N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride(BopCl),1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP), or3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP).

The example of achiral H-phosphonate moiety is a compound shown in theabove Scheme. DBU represents 1,8-diazabicyclo[5.4.0]undec-7-ene. H⁺DBUmay be, for example, ammonium ion, alkylammonium ion, heteroaromaticiminium ion, or heterocyclic iminium ion, any of which is primary,secondary, tertiary or quaternary, or a monovalent metal ion.

Reacting with Chiral Reagent

After the first activation step, the activated achiral H-phosphonatemoiety reacts with a chiral reagent, which is represented by formula(Z-I) or (Z-I′), to form a chiral intermediate of formula (Z-Va),(Z-Vb), (Z-Va′), or (Z-Vb′).

Stereospecific Condensation Step

A chiral intermediate of Formula Z-Va ((Z-Vb), (Z-Va′), or (Z-Vb′)) istreated with the second activating reagent and a nucleoside to form acondensed intermediate. The nucleoside may be on solid support. Examplesof the second activating reagent are 4,5-dicyanoimidazole (DCI),4,5-dichloroimidazole, 1-phenylimidazolium triflate (PhIMT),benzimidazolium triflate (BIT), benztriazole, 3-nitro-1,2,4-triazole(NT), tetrazole, 5-ethylthiotetrazole (ETT), 5-benzylthiotetrazole(BTT), 5-(4-nitrophenyl)tetrazole, N-cyanomethylpyrrolidinium triflate(CMPT), N-cyanomethylpiperidinium triflate,N-cyanomethyldimethylammonium triflate. A chiral intermediate of FormulaZ-Va ((Z-Vb), (Z-Va′), or (Z-Vb′)) may be isolated as a monomer.Usually, the chiral intermediate of Z-Va ((Z-Vb), (Z-Va′), or (Z-Vb′))is not isolated and undergoes a reaction in the same pot with anucleoside or modified nucleoside to provide a chiral phosphitecompound, a condensed intermediate. In other embodiments, when themethod is performed via solid phase synthesis, the solid supportcomprising the compound is filtered away from side products, impurities,and/or reagents.

Capping Step

If the final nucleic acid is larger than a dimer, the unreacted —OHmoiety is capped with a blocking group and the chiral auxiliary in thecompound may also be capped with a blocking group to form a cappedcondensed intermediate. If the final nucleic acid is a dimer, then thecapping step is not necessary.

Modifying Step

The compound is modified by reaction with an electrophile. The cappedcondensed intermediate may be executed modifying step. In someembodiments, the modifying step is performed using a sulfurelectrophile, a selenium electrophile or a boronating agent. Examples ofmodifying steps are step of oxidation and sulfurization.

In some embodiments of the method, the sulfur electrophile is a compoundhaving one of the following formulas:

S₈ (Formula Z-B), Z^(z1)—S—S—Z^(z2), or Z^(z1)—S—V^(z)—Z^(z2);

wherein Z^(z1) and Z^(z2) are independently alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, orthiocarbonyl, or Z^(z1) and Z^(z2) are taken together to form a 3 to 8membered alicyclic or heterocyclic ring, which may be substituted orunsubstituted; V^(z) is SO₂, O, or NR^(f); and R^(f) is hydrogen, alkyl,alkenyl, alkynyl, or aryl.

In some embodiments of the method, the sulfur electrophile is a compoundof following Formulae Z-A, Z-B, Z-C, Z-D, Z-E, or Z-F:

In some embodiments, the selenium electrophile is a compound having oneof the following formulae:

Se (Formula Z-G), Z³—Se—Se—Z^(z4), or Z^(z3)—Se—V—Z^(z4);

wherein Z^(z3) and Z^(z4) are independently alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, orthiocarbonyl, or Z^(z3) and Z^(z4) are taken together to form a 3 to 8membered alicyclic or heterocyclic ring, which may be substituted orunsubstituted; V^(z) is SO₂, S, O, or NR^(f); and R^(f) is hydrogen,alkyl, alkenyl, alkynyl, or aryl.

In some embodiments, the selenium electrophile is a compound of FormulaZ-G, Z-H, Z-I, Z-J, Z-K, or Z-L.

In some embodiments, the boronating agent isborane-N,N-diisopropylethylamine (BH₃ DIPEA), borane-pyridine (BH₃ Py),borane-2-chloropyridine (BH₃ CPy), borane-aniline (BH₃ An),borane-tetrahydrofiirane (BH₃ THF), or borane-dimethylsulfide (BH₃Me₂S).

In some embodiments of the method, the modifying step is an oxidationstep. In some embodiments of the method, the modifying step is anoxidation step using similar conditions as described above in thisapplication. In some embodiments, an oxidation step is as disclosed in,e.g., JP 2010-265304 A and WO2010/064146.

Chain Elongation Cycle and De-Protection Step

The capped condensed intermediate is deblocked to remove the blockinggroup at the 5′-end of the growing nucleic acid chain to provide acompound. The compound is optionally allowed to re-enter the chainelongation cycle to form a condensed intermediate, a capped condensedintermediate, a modified capped condensed intermediate, and a5′-deprotected modified capped intermediate. Following at least oneround of chain elongation cycle, the 5′-deprotected modified cappedintermediate is further deblocked by removal of the chiral auxiliaryligand and other protecting groups for, e.g., nucleobase, modifiednucleobase, sugar and modified sugar protecting groups, to provide anucleic acid. In other embodiments, the nucleoside comprising a 5′-OHmoiety is an intermediate from a previous chain elongation cycle asdescribed herein. In yet other embodiments, the nucleoside comprising a5′-OH moiety is an intermediate obtained from another known nucleic acidsynthetic method. In embodiments where a solid support is used, thephosphorus-atom modified nucleic acid is then cleaved from the solidsupport. In certain embodiments, the nucleic acids is left attached onthe solid support for purification purposes and then cleaved from thesolid support following purification.

In yet other embodiments, the nucleoside comprising a 5′-OH moiety is anintermediate obtained from another known nucleic acid synthetic method.In yet other embodiments, the nucleoside comprising a 5′-OH moiety is anintermediate obtained from another known nucleic acid synthetic methodas described in this application. In yet other embodiments, thenucleoside comprising a 5′-OH moiety is an intermediate obtained fromanother known nucleic acid synthetic method comprising one or morecycles illustrated in Scheme I. In yet other embodiments, the nucleosidecomprising a 5′-OH moiety is an intermediate obtained from another knownnucleic acid synthetic method comprising one or more cycles illustratedin Scheme I-b, I-c or I-d.

In some embodiments, the present invention provides oligonucleotidesynthesis methods that use stable and commercially available materialsas starting materials. In some embodiments, the present inventionprovides oligonucleotide synthesis methods to produce stereocontrolledphosphorus atom-modified oligonucleotide derivatives using an achiralstarting material.

In some embodiments, the method of the present invention does not causedegradations under the de-protection steps. Further the method does notrequire special capping agents to produce phosphorus atom-modifiedoligonucleotide derivatives.

In some embodiments, the present invention provides methods for thesynthesis of stereocontrolled phosphorus atom-modified oligonucleotidederivatives using a chiral monomer. In some embodiments, the first stepis reacting a nucleoside 3′-phosphoramidite derivative which isrepresented by formula (Z-Va), (Z-Vb), (Z-Va′), or (Z-Vb′) with thesecond activating reagent and a nucleoside to form a condensedintermediate. The second step is converting the condensed intermediateto the nucleic acid comprising a chiral X-phosphonate moiety. Anexemplary method is illustrated Scheme Z-2 below.

The detailed conditions of the Scheme Z-2 are similar to that of SchemeZ-1. The starting material of formula Z-Va (Z-Vb), especially of formulaZ-Va′ (or Z-Vb′), is chemically stable. As shown in a working example,the method of the present invention does not cause degradations underthe de-protection steps. Further the method does not require specialcapping agents to produce phosphorus atom-modified oligonucleotidederivatives.

In some embodiments, mechanism for the removal of auxiliaries is shownas illustrated in Scheme Z-3, below.

In Scheme Z-3, Nu stands for is a nucleophile. In some embodiments, themechanism in Scheme Z-3 is thought to be different from the previousmechanism for the removal of auxiliaries.

In some embodiments, the present invention provides a chiral reagent ora salt thereof, the chiral reagent having following chemical formula(Z-I):

wherein G^(z1) and G^(z2) are independently a hydrogen atom, a nitrogroup, a halogen atom, a cyano group, a group of formula (Z-II) or(Z-III), or both G^(z1) and G^(z2) taken together to form a group offormula (Z-IV),

wherein G²¹ to G²³ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group,

wherein G³¹ to C³³ are independently C₁₋₄ alkyl group, C₁₋₄ alkoxygroup, C₆₋₁₄ aryl group, C₇₋₁₄ aralkyl group, C₁₋₄ alkyl C₆₋₁₄ arylgroup, C₁₋₄ alkoxy C₆₋₁₄ aryl group, or C₆₋₁₄ aryl C₁₋₄ alkyl group,

wherein G⁴¹ to G⁴⁶ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group,G^(z3) and G^(z4) are independently a hydrogen atom, C₁₋₃ alkyl group,C₆₋₁₄ aryl group, or both G^(z3) and G^(z4) taken together to form aheteroatom-containing ring that has 3 to 16 carbon atoms.

In some embodiments, the present invention provides a chiral reagent, ora salt thereof, of formula Z-1 has following chemical formula (Z-I′)

wherein each variable is independently as defined above and describedherein. In some embodiments, the present invention provides a chiralreagent, or a salt thereof, of formula (Z-1′), wherein each of G^(z1)and G^(z2) is a group of formula (Z-II), wherein G²¹ to G²³ areindependently a hydrogen atom, a nitro group, a halogen atom, a cyanogroup or C₁₋₃ alkyl group. In some embodiments, the present inventionprovides a chiral reagent, or a salt thereof, of formula (Z-1′), whereineach of G^(z1) and G^(z2) is a group of formula (Z-II), wherein each ofG²¹ to G²³ is a hydrogen atom. In some embodiments, the presentinvention provides a chiral reagent, or a salt thereof, of formula(Z-1′), wherein G^(z1) is a hydrogen atom, and G^(z2) is a group offormula (Z-II), wherein G²¹ to G²³ are independently a hydrogen atom, anitro group, a halogen atom, a cyano group or C₁₋₃ alkyl group. In someembodiments, the present invention provides a chiral reagent, or a saltthereof, of formula (Z-1′), wherein G^(z1) is a hydrogen atom, andG^(z2) is a group of formula (Z-II), wherein each of G²¹ and G²² is ahydrogen atom and G²³ is a nitro group. In some embodiments, the presentinvention provides a chiral reagent, or a salt thereof, of formula(Z-1′), wherein G^(z1) is a hydrogen atom, and G^(z2) is a group offormula (Z-III), wherein G³¹ to G³³ are independently C₁₋₄ alkyl group,C₆₋₁₄ aryl group, C₇₋₁₄ aralkyl group, C₁₋₄ alkyl C₆₋₁₄ aryl group, C₁₋₄alkoxy C₆₋₁₄ aryl group, or C₆₋₁₄ aryl C₁₋₄ alkyl group. In someembodiments, the present invention provides a chiral reagent, or a saltthereof, of formula (Z-1′), wherein G^(z1) is a hydrogen atom, andG^(z2) is a group of formula (Z-III), wherein G³¹ to G³³ areindependently C₁₋₄ alkyl group, C₆ aryl group, C₇₋₁₀ aralkyl group, C₁₋₄alkyl C₆ aryl group, C₁₋₄ alkoxy C₆ aryl group, or C₆ aryl C₁₋₄ alkylgroup. In some embodiments, the present invention provides a chiralreagent, or a salt thereof, of formula (Z-1′), wherein G^(z1) is ahydrogen atom, and G^(z2) is a group of formula (Z-III), wherein G³¹ toG³³ are independently C₁₋₄ alkyl group, or C₆ aryl group. In someembodiments, the present invention provides a chiral reagent, or a saltthereof, of formula (Z-1′), wherein G^(z1) is a hydrogen atom, andG^(z2) is a group of formula (Z-III), wherein G³¹ and G³³ are C₆ arylgroup and G³² is C₁₋₂ alkyl group. In some embodiments, the presentinvention provides a chiral reagent, or a salt thereof, of formula(Z-1′), wherein G^(z1) is a hydrogen atom, and G^(z2) is a group offormula (Z-III), wherein G³¹ to G³³ are independently C₁₋₄ alkyl group.In some embodiments, the present invention provides a chiral reagent, ora salt thereof, of formula (Z-1′), wherein G^(z1) is a hydrogen atom,and G^(z2) is a group of formula (Z-III), wherein G³¹ and G³³ are C₆aryl group and G³² is C₁₋₄ alkyl group. In some embodiments, the presentinvention provides a chiral reagent, or a salt thereof, of formula(Z-1′), wherein G^(z1) is a hydrogen atom, and G^(z2) is a group offormula (Z-IV), wherein G⁴¹ to G⁴⁶ are independently a hydrogen atom, anitro group, a halogen atom, a cyano group or C₁₋₃ alkyl group. In someembodiments, the present invention provides a chiral reagent, or a saltthereof, of formula (Z-1′), wherein G^(z1) and G^(z2) are taken togetherto form a group of formula (Z-IV), wherein G⁴¹ to G⁴⁶ are independentlya hydrogen atom, a nitro group, a halogen atom, a cyano group or C₁₋₃alkyl group. In some embodiments, the present invention provides achiral reagent, or a salt thereof, of formula (Z-1′), wherein G^(z1) andG^(z2) are taken together to form a group of formula (Z-IV), whereineach of G⁴¹ to G⁴⁶ is a hydrogen atom.

In some embodiments, a chiral reagent or a salt thereof is selected fromformulae 3a, 3b, 5a, Z-5b, 7a, 7b, 9a, 9b, 11a and 11b.

In some embodiments, the present invention provides a nucleoside3′-phosphoramidite derivative which is represented by formula Z-Va orZ-Vb:

wherein G^(z1) and G^(z2) are independently a hydrogen atom, a nitrogroup, a halogen atom, a cyano group, a group of formula (Z-II) or(Z-III), or both G^(z1) and G^(z2) taken together to form a group offormula (Z-IV),

wherein G²¹ to G²³ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group,

wherein G³¹ to G³³ are independently C₁₋₄ alkyl group, C₁₋₄ alkoxygroup, C₆₋₁₄ aryl group, C₇₋₁₄ aralkyl group, C₁₋₄ alkyl C₆₋₁₄ arylgroup, C₁₋₄ alkoxy C₆₋₁₄ aryl group, or C₆₋₁₄ aryl C₁₋₄ alkyl group,

wherein G⁴¹ to G⁴⁶ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group;G^(z3) and G^(z4) are independently a hydrogen atom, C₁₋₃ alkyl group,C₆₋₁₄ aryl group, or both G^(z3) and G^(z4) taken together to form aheteroatom-containing ring that has 3 to 16 carbon atoms;G^(z5) is a protective group of a hydroxyl group;R^(z2) is independently hydrogen, —OH, —SH, —NR^(d)R^(d), —N₃, halogen,alkyl, alkenyl, alkynyl, alkyl-Y¹—, alkenyl-Y¹—, alkynyl-Y¹—, aryl-Y¹—,heteroaryl-Y¹—, —OR^(b), or —SR^(b), wherein R^(b) is a blocking moiety;Y¹ is O, NR^(d), S, or Se;R^(d) is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,substituted silyl, carbamate, —P(O)(R^(e))₂, or —HP(O)(R^(e));R^(e) is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, or heteroaryl-Y²—, or acation which is Na⁺, Li⁺, or K⁺, or —O⁻;Y² is O, NR^(d), or S;R^(z3) is a group represented by —CH₂—, —(CH₂)₂—, —CH₂NH—, or—CH₂N(CH₃)—; andBs is a group selected from the groups represented by following formula(Z-VI) to (Z-XI) or derivatives thereof.

In some embodiments, the present invention provides a nucleoside3′-phosphoramidite derivative of formula Z-Va or Z-Vb, having thestructure of (Z-Va′) or (Z-Vb′):

wherein each variable is independently as defined above and describedherein.

In some embodiments, the present invention provides a nucleoside3′-phosphoramidite derivative selected from formulae 12a, 12b, 13a, 13b,14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b, 20a, 20b,21a, 21b, 22a, 22b, 23a, 23b, 24a, 24b, 25a, 25b, 26a, 26b, 27a, 27b,28a, 28b, 29a, 29b, 30a, 30b, 31a, 31b, 32a, 32b, 33a, 33b, 34a, 34b and35a. In some embodiments, the present invention provides a nucleoside3′-phosphoramidite derivative selected from formulae 12a, 12b, 13a, 13b,14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b, 20a, 20b,21a, 21b, 22a, 22b, 23a, 23b, 24a, 24b, 25a, 25b, 26a, or 26b.

In some embodiments, the present invention provides a method forsynthesis of stereocontrolled phosphorus atom-modified oligonucleotidederivatives comprising steps of:

reacting a molecule comprising an achiral H-phosphonate moiety, a chiralreagent or a salt thereof to form a monomer of a nucleoside3′-phosphoramidite derivative;reacting the monomer and a nucleoside to form a condensed intermediate;andconverting the condensed intermediate to the nucleic acid comprising achiral X-phosphonate moiety;wherein the chiral reagent has following chemical formula (Z-I).

In some embodiments, the present invention provides a method forsynthesis of stereocontrolled phosphorus atom-modified oligonucleotidederivatives comprising steps of:

reacting a molecule comprising an achiral H-phosphonate moiety, a chiralreagent or a salt thereof to form a monomer of a nucleoside3′-phosphoramidite derivative;reacting the monomer and a nucleoside to form a condensed intermediate;andconverting the condensed intermediate to the nucleic acid comprising achiral X-phosphonate moiety;wherein the chiral reagent has following chemical formula (Z-I′).

In some embodiments, the present invention provides a method forsynthesis of stereocontrolled phosphorus atom-modified oligonucleotidederivatives comprising steps of:

reacting a molecule comprising an achiral H-phosphonate moiety, a chiralreagent or a salt thereof to form a monomer of a nucleoside3′-phosphoramidite derivative;reacting the monomer and a nucleoside to form a condensed intermediate;andconverting the condensed intermediate to the nucleic acid comprising achiral X-phosphonate moiety;wherein the chiral reagent is selected from formulae 3a, 3b, 5a, Z-5b,7a, 7b, 9a, 9b, 11a and 11b.

In some embodiments, the present invention provides a method forsynthesis of stereocontrolled phosphorus atom-modified oligonucleotidederivatives comprising steps of:

reacting a nucleoside 3′-phosphoramidite derivative which is representedby formula (Z-Va) or (Z-Vb), with an activating reagent and a nucleosideto form a condensed intermediate; and converting the condensedintermediate to the nucleic acid comprising a chiral X-phosphonatemoiety.

In some embodiments, the present invention provides a method forsynthesis of stereocontrolled phosphorus atom-modified oligonucleotidederivatives comprising steps of:

reacting a nucleoside 3′-phosphoramidite derivative represented byformula (Z-Va) or (Z-Vb), with an activating reagent and a nucleoside toform a condensed intermediate; and converting the condensed intermediateto the nucleic acid comprising a chiral X-phosphonate moiety; andwherein the nucleoside 3′-phosphoramidite derivative represented byformula (Z-Va) or (Z-Vb) is selected from formulae 12a, 12b, 13a, 13b,14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b, 20a, 20b,21a, 21b, 22a, 22b, 23a, 23b, 24a, 24b, 25a, 25b, 26a, 26b, 27a, 27b,28a, 28b, 29a, 29b, 30a, 30b, 31a, 31b, 32a, 32b, 33a, 33b, 34a, 34b and35a. In some embodiments, the present invention provides a method forsynthesis of stereocontrolled phosphorus atom-modified oligonucleotidederivatives comprising steps of:reacting a nucleoside 3′-phosphoramidite derivative represented byformula (Z-Va) or (Z-Vb), with an activating reagent and a nucleoside toform a condensed intermediate; and converting the condensed intermediateto the nucleic acid comprising a chiral X-phosphonate moiety; andwherein the nucleoside 3′-phosphoramidite derivative represented byformula (Z-Va) or (Z-Vb) is selected from formulae 12a, 12b, 13a, 13b,14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b, 20a, 20b,21a, 21b, 22a, 22b, 23a, 23b, 24a, 24b, 25a, 25b, 26a, and 26b.

Preparation and Use of Certain Chiral Auxiliaries of Formula Z-IAbbreviation

ac: acetylbz: benzoylCSO: (1S)-(+)-(10-camphorsulfonyl)oxaziridineDBU: 1,8-diazabicyclo[5.4.0]undec-7-eneDCA: dichloroacetic acidDCM: dichloromethane, CH₂Cl₂Tr: trityl, triphenylmethyl

MeIm: N-methylimidazole NIS: N-iodosuccinimide

pac: phenoxyacetylPh: phenylPhIMT: N-phenylimidazolium triflatePOS: 3-phenyl-1,2,4-dithiazoline-5-oneTBS: tert-butyldimethylsilylTBDPS: tert-butyldiphenylsilylTOM: triisopropylsiloxymethylTFA: trifluoroacetic acid

General Procedure for the Synthesis of Chirally ControlledOligonucleotides-1.

The automated solid-phase synthesis of chirally controlledoligonucleotides was performed according to the cycles shown in TableZ-1.

TABLE Z-1 Synthesis procedure. step operation reagents and solventvolume waiting time 1 detritylation 3% DCA/DCM 1.6 mL 20 s 2 coupling0.1M monomer/MeCN + 1M PhIMT 0.5 mL 5 min 3 capping Ac₂O/THF-pyridine +16% MeIm/THF 0.5 mL 30 s 4 oxidation/sulfurization 0.5M CSO/MeCN or 0.1MPOS/MeCN 0.5 mL 90 s

General Procedure for the Synthesis of Chirally ControlledOligonucleotides-2.

The automated solid-phase synthesis of chirally controlledoligonucleotides was performed according to the cycles shown in TableZ-2.

TABLE Z-2 step operation reagents and solvent volume waiting time 1detritylation 3% DCA/DCM 1.6 mL 20 s 2 coupling pre-activated monomer* +1M PhIMT 0.5 mL 5 min 3 capping Ac₂O/THF-pyridine + 16% MeIm/THF 0.5 mL30 s 4 oxidation/sulfurization 0.5M CSO/MeCN or 0.1M POS/MeCN 0.5 mL 90s *preparation of pre-activated monomer in Step 2 of TableZ- 2:

Nucleoside-3′-H-phosphonate monoester is dried by repeatedcoevaporations with dry toluene and then dissolved in dry MeCN. Ph₃PCl₂is added to the solution, and the mixture is stirred for 5 min. To themixture, a solution of chiral reagent, which is repeated coevaportionswith dry toluene and dissolved in dry MeCN, is added dropwise viasyringe, and the mixture is stirred for 5 min under argon.

After the synthesis, the resin was treated with a 25% NH₃ aqueoussolution (1 mL) for 12 h at 55° C. The mixture was cooled to roomtemperature and the resin was removed by membrane filtration. Thefiltrate was concentrated to dryness under reduced pressure. The residuewas dissolved in H₂O (3 mL) and analyzed by RP-UPLC-MS with a lineargradient of acetonitrile (0-50%/30 min) in 0.1 M triethylammoniumacetate buffer (pH 7.0) at 50° C. at a rate of 0.3 mL/min.

Example Z-1 (S)-1-Tritylpyrrolidin-2-carbaldehyde (1a)

Compound 1a was synthesized from L-proline according to the proceduredescribed in the literature (Guga, P. Curr. Top. Med. Chem. 2007, 7,695-713).

(R)-1-Tritylpyrrolidin-2-carbaldehyde (1b)

Compound 1b was synthesized from D-proline in a similar manner tocompound 1a.

(S)-2-(Methyldiphenylsilyl)-1-((S)-1-tritylpyrrolidin-2-yl)ethanol (2a)

To a solution of methyldiphenylsilylmethyl magnesium chloride in THFprepared from chloromethyldiphenylmethylsilane (4.02 g, 16.3 mmol) andmagnesium (402 mg, 16.3 mmol) in THF (14 mL) was added 1a (2.79 g, 8.14mmol) in THF (30 mL) solution with ice cooling. After stirring for 1.5 hwith ice cooling, the mixture warmed to room temperature and continuedstirring for 30 min. Saturated aqueous NH₄Cl (100 mL) was added to thereaction mixture at 0° C., and extraction was performed withdiethylether (100 mL) for three times. The combined extract was driedover Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was chromatographed on silica gel afforded 2a as a colorlessfoam (3.91 g, 87%). ¹H NMR (300 MHz, CDCl₃) δ 7.48-7.08 (25H, m),4.33-4.23 (1H, m), 3.16-2.89 (3H, m), 2.84 (1H, brs), 1.70-1.54 (1H, m),1.35 (1H, dd, J=14.7, 6.3 Hz), 1.10 (1H, dd, J=14.7, 8.1 Hz), 1.18-1.05(1H, m), 1.04-0.90 (1H, m), 0.34 (3H, s), −0.17-−0.36 (1H, m).

(S)-2-(Methyldiphenylsilyl)-1-((S)-1-pyrrolidin-2-yl)ethanol (3a)

2a (3.91 g, 7.06 mmol) was dissolved in 3% DCA in DCM (70 mL), andstirred for 10 min at room temperature. To the mixture, 1M NaOH (200 mL)was added, and extraction was performed with DCM (100 mL) for threetimes. The combined extract was dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was chromatographed onsilica gel afforded 3a as a light yellow oil (1.99 g, 90%). ¹H NMR (300MHz, CDCl₃) δ 7.57-7.52 (5H, m), 7.38-7.33 (5H, m), 3.77 (1H, ddd,J=8.9, 5.4, 3.5 Hz), 3.01 (1H, dt, J=7.4, 3.6 Hz), 2.97-2.79 (2H, m),2.27 (2H, brs), 1.76-1.53 (4H, m), 1.38 (1H, dd, J=15.0, 9.0 Hz), 1.24(1H, dd, J=15.0, 5.4 Hz), 0.65 (3H, s); ¹³C NMR (100.4 MHz, CDCl₃) δ137.4, 137.1, 134.6, 134.5, 129.1, 127.8, 69.5, 64.1, 47.0, 25.8, 24.0,19.6, −3.4. MALDI TOF-MS m/z Calcd for C₁₉H₂₆NOSi [M+H]⁺ 312.18, found312.06.

Example Z-2(R)-2-(Methyldiphenylsilyl)-1-((R)-1-tritylpyrrolidin-2-yl)ethanol (2b)

Compound 2b was obtained by using 1b instead of 1a in a similar mannerto compound 2a. ¹H NMR (300 MHz, CDCl₃) δ 7.48-7.12 (25H, m), 4.33-4.24(1H, m), 3.16-2.89 (3H, m), 2.86 (1H, brs), 1.69-1.52 (1H, m), 1.35 (1H,dd, J=14.4, 6.0 Hz), 1.10 (1H, dd, J=14.4, 8.4 Hz), 1.18-1.05 (1H, m),1.03-0.89 (1H, m), 0.33 (3H, s), −0.19-−0.39 (1H, m); ¹³C NMR (75.5 MHz,CDCl₃) δ 144.5, 137.5, 136.8, 134.6, 134.3, 129.8, 129.0, 127.8, 127.7,127.4, 126.1, 77.9, 71.7, 65.1, 53.5, 25.0, 24.8, 19.6, −4.0. MALDITOF-MS m/z Calcd for C₃₈H₄₀NOSi [M+H]⁺ 554.29, found 554.09.

(R)-2-(Methyldiphenylsilyl)-1-((R)-1-pyrrolidin-2-yl)ethanol (3b)

Compound 3b was obtained by using 2b instead of 2a in a similar mannerto compound 3a.

¹H NMR (300 MHz, CDCl₃) δ 7.58-7.52 (5H, m), 7.38-7.33 (5H, m), 3.78(1H, ddd, J=9.0, 5.1, 3.6 Hz), 3.00 (1H, dt, J=7.4, 3.3 Hz), 2.97-2.78(2H, m), 2.19 (2H, brs), 1.76-1.53 (4H, m), 1.38 (1H, dd, J=14.6, 9.0Hz), 1.24 (1H, dd, J=14.6, 5.1 Hz), 0.66 (3H, s); ¹³C NMR (75.5 MHz,CDCl₃) δ 137.5, 137.1, 134.5, 134.4, 129.0, 127.7, 69.2, 64.2, 46.9,25.8, 24.0, 19.7, -3.4. MALDI TOF-MS m/z Calcd for C₁₉H₂₆NOSi [M+H]⁺312.18, found 312.09.

Example Z-3(S)-2-(Trimethylsilyl)-1-((S)-1-tritylpyrrolidin-2-yl)ethanol (4a)

Compound 4a was obtained by using “chloromethyltrimethylsilane” insteadof “chloromethyldiphenylmethylsilane” in a similar manner to compound2a. ¹H NMR (300 MHz, CDCl₃) δ 7.58-7.51 (5H, m), 7.31-7.14 (10H, m),4.13 (1H, dt, J=7.5, 3.0 Hz), 3.39-3.31 (1H, m), 3.20-2.99 (2H, m), 2.84(1H, s), 1.74-1.57 (1H, m), 1.29-1.10 (2H, m), 0.74 (1H, dd, J=14.4, 7.2Hz), 0.46 (1H, dd, J=14.4, 7.2 Hz), -0.15 (9H, s). MALDI TOF-MS m/zCalcd for C₂₈H₃₆NOSi [M+H]⁺ 430.26, found 430.09.

(S)-2-(Trimethylsilyl)-1-((S)-1-pyrrolidin-2-yl)ethanol (5a)

Compound 5a was obtained by using 4a instead of 2a in a similar mannerto compound 3a. ¹H NMR (300 MHz, CDCl₃) δ 3.76 (1H, ddd, J=8.8, 5.7, 3.3Hz), 3.08 (1H, dt, J=7.8, 3.3 Hz), 3.02-2.87 (2H, m), 2.48 (2H, brs),1.81-1.58 (4H, m), 0.83 (1H, dd, J=14.7, 8.7 Hz), 0.68 (1H, dd, J=14.7,6.0 Hz), 0.05 (9H, s); ¹³C NMR (75.5 MHz, CDCl₃) δ 69.6, 64.3, 46.9,25.8, 23.9, 22.0, −0.8. MALDI TOF-MS m/z Calcd for C₉H₂₂NOSi [M+H]⁺188.15, found 188.00.

Example Z-5 (R)-2,2-Diphenyl-1-((S)-1-tritylpyrrolidin-2-yl)ethanol (6a)

To a solution of diphenylmethane (6.7 mL, 40 mmol) in anhydrous THF (36mL), n-BuLi (1.67M solution of Hexane, 24 mL, 40 mmol) was addeddropwise at room temperature and stirred for 1 h. To the mixture, 1a(3.41 g, 10 mmol), which was dried by repeated coevaporations withtoluene, in anhydrous THF (40 mL) was slowly added at 0° C., andcontinued stirring for 45 min. A saturated NH₄Cl aqueous solution (100mL) and Et₂O (100 mL) were then added, and the organic layer wasseparated and the aqueous layer was extracted with Et₂O (2×100 mL). Theorganic layer were combined, dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified bychromatography on silica gel to afford 6a (1.41 g, 28%) as white foam.

(R)-2,2-Diphenyl-1-((S)-pyrrolidin-2-yl)ethanol (7a)

6a (650 mg, 1.27 mmol) was dissolved in 3% DCA in DCM (13 mL), andstirred for 10 min at room temperature. To the mixture, 1M NaOH (40 mL)was added, and extraction was performed with DCM (30 mL) for threetimes. The combined extract was dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was chromatographed onsilica gel afforded 7a as a light yellow oil (316 mg, 93%). ¹H NMR (300MHz, CDCl₃) δ 7.44-7.38 (2H, m), 7.33-7.14 (8H, m), 4.46 (1H, dd, J=9.9,3.3 Hz), 3.91 (1H, d, J=9.9 Hz), 3.02-2.88 (2H, m), 2.81-2.69 (1H, m),2.52 (2H, brs), 1.88-1.56 (4H, m); ¹³C NMR (75.5 MHz, CDCl₃) δ 142.3,142.0, 128.6, 128.5, 128.4, 128.2, 126.5, 126.4, 73.5, 60.1, 55.8, 46.6,25.8, 23.4. MALDI TOF-MS m/z Calcd for C₁₈H₂₂NO [M+H]⁺ 268.17, found268.06.

Example Z-6 (S)-2,2-Diphenyl-1-((R)-1-tritylpyrrolidin-2-yl)ethanol (6b)

Compound 6b was obtained by using 1b instead of 1a in a similar mannerto compound 6a. ¹H NMR (300 MHz, CDCl₃) δ 7.44-7.37 (6H, m), 7.30-7.01(17H, m), 6.66-6.61 (2H, m), 4.80 (1H, d, J=10.8 Hz), 3.63 (1H, d,J=10.8 Hz), 3.36-3.28 (1H, m), 3.22-3.09 (1H, m), 3.01-2.89 (1H, m),2.66 (1H, s), 1.90-1.75 (1H, m), 1.29-1.04 (2H, m), 0.00-0.19 (1H, m);¹³C NMR (75.5 MHz, CDCl₃) δ 144.2, 142.9, 141.6, 130.0, 128.5, 128.4,127.9, 127.8, 127.4, 126.4, 126.2, 77.9, 75.9, 61.9, 55.4, 53.4, 24.7,24.5. MALDI TOF-MS m/z Calcd for C₃₇H₃₆NO [M+H]⁺ 510.28, found 510.11.

(S)-2,2-Diphenyl-1-((R)-pyrrolidin-2-yl)ethanol (7b)

Compound 7b was obtained by using 6b instead of 6a in a similar mannerto compound 7a. ¹H NMR (300 MHz, CDCl₃) δ 7.45-7.14 (10H, m), 4.45 (1H,dd, J=9.9, 3.3 Hz), 3.91 (1H, d, J=9.9 Hz), 3.00-2.89 (2H, m), 2.82-2.71(1H, m), 2.40 (2H, brs), 1.87-1.55 (4H, m); ¹³C NMR (75.5 MHz, CDCl₃) δ142.3, 142.0, 128.5, 128.3, 128.1, 126.3, 126.2, 73.4, 60.1, 55.9, 46.5,25.8, 23.5. MALDI TOF-MS m/z Calcd for C₁₈H₂₂NO [M+H]⁺ 268.17, found268.03.

Example Z-7 (R)-2-(4-Nitrophenyl)-1-((S)-1-tritylpyrrolidin-2-yl)ethanol(8a)

Compound 8a was obtained by using “4-nitrobenzylchloride” instead of“diphenylmethane” in a similar manner to compound 6a. ¹H NMR (300 MHz,CDCl₃) δ 8.09-8.03 (2H, m), 7.49-7.43 (6H, m), 7.28-7.09 (11H, m), 4.23(1H, ddd, J=8.3, 5.6, 3.0 Hz), 3.43-3.33 (1H, m), 3.23-3.11 (1H, m),3.07-2.96 (1H, m), 2.83 (1H, brs), 2.74 (1H, dd, J=13.8, 8.4 Hz), 2.49(1H, dd, J=13.8, 5.1 Hz), 1.83-1.67 (1H, m), 1.41-1.17 (2H, m),0.27-0.08 (1H, m); ¹³C NMR (75.5 MHz, CDCl₃) δ 147.3, 146.3, 144.3,129.8, 129.6, 127.5, 126.3, 123.4, 77.9, 74.8, 63.5, 53.2, 39.5, 25.0,24.9. MALDI TOF-MS m/z Calcd for C₃₁H₃₁N₂O₃ [M+H]⁺ 479.23, found 479.08.

(R)-2-(4-Nitrophenyl)-1-((S)-pyrrolidin-2-yl)ethanol (9a)

Compound 9a was obtained by using 8a instead of 6a in a similar mannerto compound 7a. ¹H NMR (300 MHz, CDCl₃) δ 8.15 (2H, d, J=8.7 Hz), 7.42(2H, d, J=8.7 Hz), 3.86-3.79 (1H, m), 3.16-3.07 (1H, m), 2.99-2.68 (6H,m), 1.84-1.68 (4H, m); ¹³C NMR (75.5 MHz, CDCl₃) δ 147.4, 146.2, 129.9,123.2, 72.4, 62.0, 46.6, 40.4, 25.7, 24.4. MALDI TOF-MS m/z Calcd forC₁₂H₁₇N₂O₃ [M+H]⁺ 237.12, found 237.01.

Example Z-8 (S)-2-(4-Nitrophenyl)-1-((R)-1-tritylpyrrolidin-2-yl)ethanol(8b)

Compound 8b was obtained by using 1b instead of 1a in a similar mannerto compound 8a. ¹H NMR (300 MHz, CDCl₃) δ 8.09-8.04 (2H, m), 7.49-7.43(6H, m), 7.28-7.09 (11H, m), 4.22 (1H, ddd, J=8.4, 5.6, 3.0 Hz),3.43-3.33 (1H, m), 3.24-3.10 (1H, m), 3.08-2.94 (1H, m), 2.81 (1H, brs),2.75 (1H, dd, J=14.0, 8.1 Hz), 2.49 (1H, dd, J=14.0, 5.1 Hz), 1.81-1.67(1H, m), 1.40-1.16 (2H, m), 0.26-0.09 (1H, m); ¹³C NMR (75.5 MHz, CDCl₃)δ 147.3, 144.3, 129.8, 129.6, 129.4, 126.3, 123.5, 77.9, 74.8, 63.5,53.2, 39.5, 25.0, 24.9. MALDI TOF-MS m/z Calcd for C₃₁H₃₁N₂O₃ [M+H]⁺479.23, found 479.08.

(S)-2-(4-Nitrophenyl)-1-((R)-pyrrolidin-2-yl)ethanol (9b)

Compound 9b was obtained by using 8b instead of 8a in a similar mannerto compound 9a. ¹H NMR (300 MHz, CDCl₃) δ 8.19-8.13 (2H, m), 7.45-7.39(2H, m), 3.83 (1H, ddd, J=7.7, 5.4, 3.9 Hz), 3.14 (1H, dt, J=7.7, 3.9Hz), 3.01-2.87 (2H, m), 2.83 (1H, d, J=3.3 Hz), 2.81 (1H, s), 2.62 (2H,brs), 1.79-1.72 (4H, m); ¹³C NMR (75.5 MHz, CDCl₃) δ 147.3, 146.5,130.0, 123.5, 72.7, 61.7, 46.7, 40.1, 25.8, 24.2. MALDI TOF-MS m/z Calcdfor C₁₂H₁₇N₂O₃ [M+H]⁺ 237.12, found 237.02.

Example Z-9 (R)-(9H-Fluoren-9-yl)((S)-1-tritylpyrrolidin-2-yl)methanol(10a)

Compound 10a was obtained by using “fluorene” instead of“diphenylmethane” in a similar manner to compound 6a. ¹H NMR (300 MHz,CDCl₃) δ 7.70 (1H, d, J=7.5 Hz), 7.66 (1H, d, J=7.8 Hz), 7.55 (2H, d,J=7.5 Hz), 7.44-7.09 (18H, m), 6.87-6.62 (1H, m), 4.55-4.48 (1H, m),4.06 (1H, d, J=7.5 Hz), 3.43-3.34 (1H, m), 3.18-3.06 (1H, m), 2.98-2.88(1H, m), 2.85 (1H, brs), 1.42-1.24 (1H, m), 1.18-1.04 (1H, m), 0.53-0.39(1H, m), −0.02-−0.20 (1H, m); MALDI TOF-MS m/z Calcd for C₃₇H₃₄NO [M+H]⁺508.26, found 508.12.

(R)-(9H-Fluororen-9-yl)((S)-pyrrolidin-2-yl)methanol (11a)

Compound 11a was obtained by using 10a instead of 6a in a similar mannerto compound 7a. ¹H NMR (300 MHz, CDCl₃) δ 7.76 (2H, d, J=7.5 Hz), 7.68(2H, t, J=8.0 Hz), 7.43-7.35 (2H, m), 7.34-7.25 (2H, m), 4.28 (1H, d,J=6.3 Hz), 4.03 (1H, dd, J=6.5, 4.2 Hz), 3.19-3.11 (1H, m), 2.97-2.88(1H, m), 2.86-2.76 (1H, m), 2.02 (2H, brs), 1.77-1.53 (3H, m), 1.38-1.23(1H, m); MALDI TOF-MS m/z Calcd for C₁₈H₂₀NO [M+H]⁺ 266.15, found266.04.

(S)-2-Tosyl-1-((S)-1-tritylpyrrolidin-2-yl)ethanol (12a′)

Compound 12a′ was obtained by using “chloromethyl p-tolyl sulfone”instead of “chloromethyldiphenylmethylsilane” in a similar manner tocompound 2a.

¹H NMR (600 MHz, CDCl₃) δ 7.66 (2H, d, J=8.4 Hz), 7.48-7.44 (6H, m),7.35 (2H, d, J=7.2 Hz), 7.21-7.13 (9H, m), 4.39-4.36 (1H, m), 3.33 (1H,s), 3.24-3.20 (1H, m), 3.19-3.10 (2H, m), 2.98-2.92 (2H, m), 2.49 (3H,s), 1.55-1.49 (1H, m), 1.33-1.26 (1H, m), 1.12-1.04 (1H, m), 0.22-0.14(1H, m); ¹³C NMR (150.9 MHz, CDCl₃) δ 144.6, 144.5, 136.3, 129.9, 129.5,128.1, 127.5, 126.2, 78.0, 69.1, 63.9, 60.2, 52.6, 25.5, 24.7, 21.7.

(S)-2-Tosyl-1-((S)-1-tritylpyrrolidin-2-yl)ethanol (13a′)

Compound 13a′ was obtained by using 12a′ instead of 2a in a similarmanner to compound 3a.

¹H NMR (600 MHz, CDCl₃) δ 7.82 (2H, d, J=8.4 Hz), 7.37 (2H, d, J=8.4Hz), 4.01 (1H, ddd, J=12.0, 5.1, 3.0 Hz), 3.32 (1H, dd, J=14.4, 3.0 Hz),3.25 (1H, dd, J=14.4, 9.0 Hz), 3.16 (1H, dt, J=7.8, 5.1 Hz), 2.90-2.82(2H, m), 2.46 (3H, s), 2.04 (2H, brs), 1.78-1.63 (3H, m), 1.62-1.55 (1H,m); ¹³C NMR (150.9 MHz, CDCl₃) δ 144.5, 136.7, 129.7, 127.7, 67.4, 61.8,60.1, 46.7, 25.7, 21.4. MALDI TOF-MS m/z Calcd for C₁₃H₂₀NO₃S [M+H]⁺270.12, found 270.04.

(R)-2-Tosyl-1-((R)-1-tritylpyrrolidin-2-yl)ethanol (12b′)

Compound 12b′ was obtained by using 1b instead of 1a in a similar mannerto compound 12a′.

¹H NMR (600 MHz, CDCl₃) δ 7.66 (2H, d, J=8.4 Hz), 7.47-7.44 (6H, m),7.35 (2H, d, J=7.8 Hz), 7.21-7.13 (9H, m), 4.37 (1H, dt, J=8.6, 2.4 Hz),3.33 (1H, s), 3.23-3.20 (1H, m), 3.19-3.12 (2H, m), 2.98-2.92 (2H, m),2.49 (3H, s), 1.56-1.49 (1H, m), 1.32-1.26 (1H, m), 1.11-1.03 (1H, m),0.23-0.15 (1H, m); ¹³C NMR (150.9 MHz, CDCl₃) δ 144.6, 144.5, 136.3,129.9, 129.6, 128.1, 127.6, 126.2, 78.0, 69.1, 63.9, 60.2, 52.6, 25.5,24.7, 21.7.

(R)-2-Tosyl-1-(R)-1-tritylpyrrolidin-2-yl)ethanol (13b′)

Compound 13b′ was obtained by using 12b′ instead of 12a′ in a similarmanner to compound 13a′.

¹H NMR (600 MHz, CDCl₃) δ 7.82 (2H, d, J=8.4 Hz), 7.37 (2H, d, J=8.4Hz), 4.01 (1H, ddd, J=9.0, 5.1, 3.0 Hz), 3.32 (1H, dd, J=14.4, 3.0 Hz),3.25 (1H, dd, J=14.4, 9.0 Hz), 3.17 (1H, dt, J=7.2, 5.1 Hz), 2.89-2.83(2H, m), 2.46 (3H, s), 2.04 (2H, brs), 1.79-1.64 (3H, m), 1.62-1.55 (1H,m); ¹³C NMR (150.9 MHz, CDCl₃) δ 144.8, 136.6, 129.8, 127.9, 67.7, 61.8,60.1, 46.8, 25.9, 25.8, 21.6. MALDI TOF-MS m/z Calcd for C₁₃H₂₀NO₃S[M+H]⁺ 270.12, found 270.05.

Example Z-10 Oxazaphospholidine Monomer 12a

3a (560 mg, 1.80 mmol) were dried by repeated coevaporations with drytoluene and dissolved in dry diethylether (0.90 mL) under argon.N-Methylmorpholine (400 micro L, 3.60 mmol) was added to the solution,and the resultant solution was added dropwise to a solution of PCl₃ (160micro L, 1.80 mmol) in dry diethylether (0.90 mL) at 0° C. under argonwith stirring. The mixture was then allowed to warm to room temperatureand stirred for 30 min. The resultant N-methylmorpholine hydrochloridewas removed by filtration under nitrogen, and the filtrate wasconcentrated to dryness under reduced pressure to afford crude2-chloro-1,3,2-oxazaphospholidine derivative. The crude materials weredissolved in freshly distilled THF (3.6 mL) to make 0.5 M solutions,which were used to synthesize the nucleoside 3′-O-oxazaphospholidineswithout further purification.

5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine (636 mg, 0.84mmol) was dried by repeated coevaporations with dry toluene, anddissolved in freshly distilled THF (2.5 mL) under argon. Et₃N (0.58 mL,4.2 mmol) was added, and the mixture was cooled to −78° C. A 0.5 Msolution of the corresponding crude 2-chloro-1,3,2-oxazaphospholidinederivative in freshly distilled THF (3.6 mL, 1.80 mmol) was addeddropwise via a syringe, and the mixture was stirred for 15 min at roomtemperature. A saturated NaHCO₃ aqueous solution (70 mL) and CHCl₃ (70mL) were then added, and the organic layer was separated and washed withsaturated NaHCO₃ aqueous solutions (2×70 mL). The combined aqueouslayers were back-extracted with CHCl₃ (70 mL). The organic layers werecombined, dried over Na₂SO₄, filtered and concentrated under reducedpressure. The residue was purified by chromatography on silica gel toafford 12a (829 mg, 90%) as a white foam. ¹H NMR (300 MHz, CDCl₃) δ 8.77(1H, brs), 7.99 (1H, s), 7.54-6.98 (24H, m), 6.81-6.73 (4H, m), 6.35(1H, dd, J=8.0, 6.3 Hz), 4.89-4.73 (411, m), 4.68 (2H, brs), 4.05-3.98(1H, m), 3.75 (6H, s), 3.62-3.46 (1H, m), 3.41-3.20 (3H, m), 3.18-3.04(1H, m), 3.08 (2H, t, J=6.6 Hz), 2.58-2.36 (2H, m), 1.94-1.59 (2H, m),1.56 (1H, dd, J=15.0, 8.7 Hz), 1.43 (1H, dd, J=15.0, 5.7 Hz), 1.33-1.16(2H, m), 0.62 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 153.5 (1P, s).

Example Z-11 Oxazaphospholidine Monomer 12b

Compound 12b was obtained by using 3b instead of 3a in a similar mannerto compound 12a. ¹H NMR (300 MHz, CDCl₃) δ 8.80 (1H, brs), 7.96 (1H, s),7.54-6.96 (24H, m), 6.79-6.71 (4H, m), 6.19 (1H, t, J=6.6 Hz), 4.90-4.73(4H, m), 4.66 (2H, brs), 4.16-4.08 (1H, m), 3.76 (6H, s), 3.60-3.36 (2H,m), 3.29 (1H, d, J=3.9 Hz), 3.27-3.12 (2H, m), 3.09 (2H, t, J=6.6 Hz),2.59-2.46 (1H, m), 2.07-1.97 (1H, m), 1.94-1.41 (5H, m), 1.36-1.18 (1H,m), 0.65 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 157.1 (1P, s).

Example Z-12 Oxazaphospholidine Monomer 13a

Compound 13a was obtained by using “5′-O-(DMTr)thymidine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a. ¹H NMR (300 MHz, CDCl₃) δ 7.58-7.23 (21H, m),6.86-6.79 (4H, m), 6.35 (1H, dd, J=8.1, 5.7 Hz), 4.79-4.67 (2H, m),3.83-3.78 (1H, m), 3.78 (6H, s), 3.59-3.43 (1H, m), 3.34 (1H, dd,J=10.5, 2.4 Hz), 3.35-3.24 (1H, m), 3.20 (1H, dd, J=10.5, 2.4 Hz),3.16-3.02 (1H, m), 2.36-2.26 (1H, m), 2.15-2.02 (1H, m), 1.92-1.77 (1H,m), 1.74-1.59 (1H, m), 1.52 (1H, dd, J=14.7, 9.0 Hz), 1.40 (3H, s),1.45-1.15 (3H, m), 0.60 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 153.7 (1P,s).

Example Z-13 Oxazaphospholidine Monomer 13b

Compound 13b was obtained by using 3b instead of 3a in a similar mannerto compound 13a. ¹H NMR (300 MHz, CDCl₃) δ 8.46 (1H, brs), 7.59-7.20(20H, m), 6.86-6.79 (4H, m), 6.26 (1H, t, J=6.8 Hz), 4.78-4.65 (2H, m),4.01-3.95 (1H, m), 3.78 (6H, s), 3.55-3.40 (1H, m), 3.42 (1H, dd,J=10.5, 2.7 Hz), 3.40-3.28 (1H, m), 3.22 (1H, dd, J=10.5, 3.0 Hz),3.19-3.06 (1H, m), 2.16-1.95 (2H, m), 1.90-1.54 (3H, m), 1.49-1.35 (1H,m), 1.43 (3H, s), 1.34-1.17 (2H, m), 0.67 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) δ 156.2 (1P, s). Oligos were synthesized using the above compound13b by the general method disclosed above.

Example Z-14 Oxazaphospholidine Monomer 14a

Compound 14a was obtained by using“5′-O-(DMTr)-4-N-(isobutyryl)cytidine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a. ¹H NMR (300 MHz, CDCl₃) δ 8.33 (1H, brs), 8.17(1H, d, J=7.5 Hz), 7.52-7.22 (19H, m), 7.07 (1H, d, J=7.5 Hz), 6.88-6.81(4H, m), 6.20 (1H, t, J=6.2 Hz), 4.81-4.64 (2H, m), 3.93-3.87 (1H, m),3.79 (6H, s), 3.59-3.43 (1H, m), 3.39-3.29 (3H, m), 3.16-3.02 (1H, m),2.69-2.52 (2H, m), 2.12-2.00 (1H, m), 1.91-1.50 (3H, m), 1.47-1.32 (2H,m), 1.27-1.16 (7H, m), 0.60 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 154.8(1P, s).

Example Z-16 Oxazaphospholidine Monomer 14b

Compound 14b was obtained by using 3b instead of 3a in a similar mannerto compound 14a. ¹H NMR (300 MHz, CDCl₃) δ 8.33 (1H, d, J=7.5 Hz), 8.23(1H, brs), 7.57-7.22 (19H, m), 7.12 (1H, d, J=7.5 Hz), 6.88-6.81 (4H,m), 6.15 (1H, dd, J=6.6, 4.2 Hz), 4.82-4.63 (2H, m), 4.03-3.97 (1H, m),3.80 (6H, s), 3.55-3.26 (4H, m), 3.19-3.05 (1H, m), 2.59 (1H, quintet,J=6.9 Hz), 2.39-2.27 (1H, m), 2.21-2.10 (1H, m), 1.90-1.56 (3H, m),1.50-1.32 (2H, m), 1.26-1.17 (7H, m), 0.66 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) δ 157.2 (1P, s).

Example Z-17 Oxazaphospholidine Monomer 15a

Compound 15a was obtained by using “5′-O-(DMTr)-6-N-(benzoyl)adenosine”instead of “5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 12a.

¹H NMR (600 MHz, CDCl₃) δ 8.71 (1H, s), 8.12 (1H, s), 8.04 (2H, d, J=7.8Hz), 7.62-7.15 (23H, m), 6.80-6.75 (4H, m), 6.37 (1H, dd, J=7.8, 6.0Hz), 4.94-4.88 (1H, m), 4.80 (1H, ddd, J=12.0, 6.0, 5.4 Hz), 4.07-4.04(1H, m), 3.76 (6H, s), 3.58-3.49 (1H, m), 3.41-3.34 (1H, m), 3.33 (1H,dd, J=10.8, 4.8 Hz), 3.25 (1H, dd, J=10.8, 4.8 Hz), 3.13-3.06 (1H, m),2.66-2.58 (1H, m), 2.40-2.35 (1H, m), 1.91-1.84 (1H, m), 1.73-1.66 (1H,m), 1.56 (1H, dd, J=15.0, 9.0 Hz), 1.44 (1H, dd, J=15.0, 5.4 Hz),1.47-1.41 (1H, m), 1.30-1.23 (1H, m), 0.63 (3H, s); ³¹P NMR (243.0 MHz,CDCl₃) δ 151.8 (1P, s).

Example Z-18 Oxazaphospholidine Monomer 15b

Compound 15b was obtained by using 3b instead of 3a in a similar mannerto compound 15a. ¹H NMR (300 MHz, CDCl₃) δ 9.06 (1H, brs), 8.76 (1H, s),8.12 (1H, s), 8.07-7.99 (2H, m), 7.64-7.14 (22H, m), 6.83-6.75 (4H, m),6.25 (1H, t, J=6.6 Hz), 4.86-4.75 (2H, m), 4.20-4.15 (1H, m), 3.77 (6H,s), 3.61-3.38 (2H, m), 3.36 (1H, dd, J=10.2, 4.2 Hz), 3.27 (1H, dd,J=10.2, 4.2 Hz), 3.27-3.13 (1H, m), 2.71-2.59 (1H, m), 2.12-2.01 (1H,m), 1.94-1.42 (5H, m), 1.36-1.20 (1H, m), 0.67 (3H, s)); ³¹P NMR (121.5MHz, CDCl₃) δ 157.3 (1P, s).

Example Z-19 Oxazaphospholidine Monomer 16a

Compound 16a was obtained by using 7a instead of 3a in a similar mannerto compound 13a. ¹H NMR (300 MHz, CDCl₃) δ 7.57 (1H, d, J=0.9 Hz),7.37-6.94 (20H, m), 6.87-6.78 (4H, m), 6.48 (1H, dd, J=8.6, 5.7 Hz),5.42 (1H, dd, J=11.0, 5.1 Hz), 4.81-4.71 (1H, m), 4.02 (1H, d, J=11.0Hz), 3.83 (1H, d, J=2.1 Hz), 3.79 (6H, s), 3.61-3.41 (2H, m), 3.24-3.09(1H, m), 3.16 (1H, dd, J=10.8, 2.4 Hz), 3.02 (1H, dd, J=10.8, 2.4 Hz),2.54-2.44 (1H, m), 2.34-2.22 (1H, m), 1.94-1.79 (1H, m), 1.74-1.56 (1H,m), 1.38 (3H, s), 1.38-1.28 (2H, m); ³¹P NMR (121.5 MHz, CDCl₃) δ 160.9(1P, s).

Example Z-20 Oxazaphospholidine Monomer 16b

Compound 16b was obtained by using 3b instead of 3a in a similar mannerto compound 16a. ¹H NMR (300 MHz, CDCl₃) δ 7.57 (1H, d, J=1.5 Hz),7.43-7.11 (20H, m), 6.85-6.78 (4H, m), 6.48 (1H, dd, J=7.5, 5.7 Hz),5.58 (1H, dd, J=11.4, 5.1 Hz), 4.82-4.73 (1H, m), 4.17-4.02 (2H, m),3.78 (6H, s), 3.56-3.40 (3H, m), 3.32 (1H, dd, J=10.7, 2.4 Hz),3.22-3.07 (1H, m), 2.26-2.04 (2H, m), 1.95-1.81 (1H, m), 1.74-1.56 (1H,m), 1.40 (3H, d, J=1.5 Hz), 1.44-1.34 (2H, m); ³¹P NMR (121.5 MHz,CDCl₃) δ 162.2 (1P, s).

Example Z-21 Oxazaphospholidine Monomer 17a

Compound 17a was obtained by using 9a instead of 3a in a similar mannerto compound 13a. ¹H NMR (300 MHz, CDCl₃) δ 9.22 (1H, brs), 8.05-7.99(2H, m), 7.52 (1H, d, J=1.2 Hz), 7.41-7.19 (11H, m), 6.87-6.79 (4H, m),6.37 (1H, dd, J=8.4, 5.7 Hz), 4.88-4.75 (2H, m), 3.86-3.80 (1H, m), 3.79(6H, d, J=1.2 Hz), 3.64-3.49 (2H, m), 3.27-3.12 (3H, m), 2.97 (2H, d,J=6.6 Hz), 2.51-2.41 (1H, m), 2.33-2.20 (1H, m), 2.03-1.75 (2H, m),1.72-1.59 (1H, m), 1.46-1.36 (1H, m), 1.40 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) δ 157.5 (1P, s).

Example Z-22 Oxazaphospholidine Monomer 17b

Compound 17b was obtained by using 9b instead of 9a in a similar mannerto compound 17a. ¹H NMR (300 MHz, CDCl₃) δ 8.67 (1H, brs), 8.18-8.11(2H, m), 7.57 (1H, d, J=1.2 Hz), 7.47-7.22 (11H, m), 6.86-6.79 (4H, m),6.29 (1H, t, J=6.6 Hz), 4.87 (1H, dt, J=7.5, 5.7 Hz), 4.80-4.72 (1H, m),4.11-4.05 (1H, m), 3.79 (6H, s), 3.67-3.47 (2H, m), 3.43 (1H, dd,J=10.8, 2.7 Hz), 3.27 (1H, dd, J=10.8, 2.4 Hz), 3.25-3.13 (1H, m),3.07-2.99 (2H, m), 2.19-2.12 (2H, m), 2.03-1.62 (3H, m), 1.46-1.30 (1H,m), 1.41 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 158.1 (1P, s).

Example Z-23 Oxazaphospholidine Monomer 18a

Compound 18a was obtained by using“5′-O-(DMTr)-2′-O-TOM-6-N-(acetyl)adenosine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a. ¹H NMR (300 MHz, CDCl₃) δ 8.82 (1H, brs), 8.49(1H, s), 8.10 (1H, s), 7.58-7.17 (19H, m), 6.83-6.73 (4H, m), 6.11 (1H,d, J=6.6 Hz), 5.15 (1H, dd, J=6.6, 5.4 Hz), 4.98-4.77 (4H, m), 4.18-4.11(1H, m), 3.76 (6H, s), 3.59-3.25 (4H, m), 3.16-3.02 (1H, m), 2.62 (3H,s), 1.91-1.53 (3H, m), 1.49-1.18 (3H, m), 0.96-0.80 (3H, m), 0.90 (18H,s), 0.62 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 156.7 (1P, s).

Example Z-24 Oxazaphospholidine Monomer 18b

Compound 18b was obtained by using 3b instead of 3a in a similar mannerto compound 18a. ¹H NMR (300 MHz, CDCl₃) δ 8.56 (1H, brs), 8.55 (1H, s),8.13 (1H, s), 7.57-7.17 (19H, m), 6.82-6.73 (4H, m), 6.16 (1H, d, J=5.7Hz), 5.06 (1H, t, J=5.6 Hz), 4.93 (1H, d, J=5.1 Hz), 4.83 (1H, d, J=5.1Hz), 4.81-4.69 (2H, m), 4.27-4.19 (1H, m), 3.76 (6H, s), 3.55-3.40 (2H,m), 3.33-3.16 (2H, m), 3.12-2.97 (1H, m), 2.63 (3H, s), 1.88-1.52 (3H,m), 1.45-1.16 (3H, m), 0.91-0.79 (3H, m), 0.86 (18H, s), 0.64 (3H, s);³¹P NMR (121.5 MHz, CDCl₃) δ 154.8 (1P, s).

Example Z-25 Oxazaphospholidine Monomer 19a

Compound 19a was obtained by using “5′-O-(DMTr)-2′-O-(methyl)uridine”instead of “5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 12a. ¹H NMR (300 MHz, CDCl₃) δ 7.91 (1H,d, J=7.8 Hz), 7.58-7.20 (19H, m), 6.88-6.80 (4H, m), 5.96 (1H, d, J=3.3Hz), 5.19 (1H, d, J=7.8 Hz), 4.88-4.78 (1H, m), 4.66-4.57 (1H, m),4.03-3.95 (1H, m), 3.90-3.74 (1H, m), 3.78 (6H, s), 3.77-3.71 (1H, m),3.58-3.29 (2H, m), 3.45 (3H, s), 3.13-2.82 (2H, m), 1.88-1.53 (3H, m),1.49-1.16 (3H, m), 0.60 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 155.3 (1P,s).

Example Z-26 Oxazaphospholidine Monomer 19b

Compound 19b was obtained by using 3b instead of 3a in a similar mannerto compound 19a. ¹H NMR (300 MHz, CDCl₃) δ 8.10 (1H, d, J=8.4 Hz),7.58-7.20 (19H, m), 6.87-6.79 (4H, m), 5.89 (1H, d, J=1.5 Hz), 5.21 (1H,d, J=8.4 Hz), 4.92-4.82 (1H, m), 4.73-4.63 (1H, m), 4.15-4.08 (1H, m),3.89-3.73 (1H, m), 3.78 (6H, s), 3.66-3.62 (1H, m), 3.57-3.27 (2H, m),3.30 (3H, s), 3.17-2.82 (2H, m), 1.89-1.55 (3H, m), 1.55-1.40 (1H, m),1.35-1.15 (2H, m), 0.66 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 157.5 (1P,s).

Example Z-27 Oxazaphospholidine Monomer 20a

Compound 20a was obtained by using“5′-O-(DMTr)-2′-deoxy-2′-fluorouridine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a. ¹H NMR (300 MHz, CDCl₃) δ 7.85 (1H, d, J=8.1Hz), 7.58-7.20 (19H, m), 6.87-6.79 (4H, m), 5.98 (1H, d, J=16.5 Hz),5.23 (1H, d, J=8.1 Hz), 4.86-4.61 (3H, m), 3.99 (1H, d, J=6.9 Hz), 3.76(6H, d, J=3.0 Hz), 3.56-3.34 (4H, m), 3.10-2.96 (1H, m), 1.88-1.74 (1H,m), 1.72-1.52 (2H, m), 1.48-1.16 (3H, m), 0.61 (3H, s); ³¹P NMR (121.5MHz, CDCl₃) δ 154.3 (1P, d, J=8.9 Hz).

Example Z-28 Oxazaphospholidine Monomer 20b

Compound 20b was obtained by using 3b instead of 3a in a similar mannerto compound 20a. ¹H NMR (300 MHz, CDCl₃) δ 8.01 (1H, d, J=8.4 Hz),7.58-7.20 (19H, m), 6.87-6.79 (4H, m), 6.03 (1H, d, J=16.2 Hz), 5.29(1H, d, J=8.4 Hz), 4.96 (1H, dd, J=13.1, 7.5 Hz), 4.80-4.54 (2H, m),4.15 (1H, d, J=9.0 Hz), 3.78 (6H, s), 3.61-3.39 (3H, m), 3.37-3.25 (1H,m), 3.23-3.09 (1H, m), 1.91-1.56 (3H, m), 1.51-1.13 (3H, m), 0.66 (3H,s); ³¹P NMR (121.5 MHz, CDCl₃) δ 158.9 (1P, d, J=4.4 Hz).

Example Z-29 Oxazaphospholidine Monomer 21a

Compound 21a was obtained by using“5′-O-(DMTr)-2′-O-methoxyethyl-5-methyluridine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a. ¹H NMR (300 MHz, CDCl₃) δ 7.62-7.18 (21H, m),6.84 (4H, d, J=8.7 Hz), 6.07 (1H, d, J=5.7 Hz), 4.86-4.76 (1H, m),4.63-4.54 (1H, m), 4.20 (1H, t, J=5.4 Hz), 3.95-3.89 (1H, m), 3.78 (6H,s), 3.78-3.71 (2H, m), 3.60-3.48 (2H, m), 3.44-3.02 (5H, m), 3.31 (3H,s), 1.88-1.15 (6H, m), 1.35 (3H, s), 0.58 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) δ 156.3 (1P, s).

Example Z-30 Oxazaphospholidine Monomer 21b

Compound 21b was obtained by using 3b instead of 3a in a similar mannerto compound 21a. ¹H NMR (300 MHz, CDCl₃) δ 7.71 (1H, d, J=1.2 Hz),7.55-7.22 (20H, m), 6.86-6.78 (4H, m), 5.99 (1H, d, J=3.9 Hz), 4.78-4.62(2H, m), 4.13-4.08 (1H, m), 4.07-4.02 (1H, m), 3.77 (6H, s), 3.77-3.70(1H, m), 3.65-3.56 (1H, m), 3.52-3.36 (4H, m), 3.33-3.14 (2H, m), 3.29(3H, s), 3.08-2.94 (1H, m), 1.86-1.72 (1H, m), 1.71-1.55 (2H, m), 1.30(3H, d, J=1.2 Hz), 1.47-1.16 (3H, m) 0.64 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) δ 155.6 (1P, s).

Example Z-31 Oxazaphospholidine Monomer 22a

Compound 22a was obtained by using“5′-O-(DMTr)-2′-O-methyl-4-N-(isobutyryl)cytidine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a. ¹H NMR (300 MHz, CDCl₃) δ 8.49 (1H, d, J=7.2Hz), 7.58-7.20 (19H, m), 6.96 (1H, d, J=7.2 Hz), 6.90-6.82 (4H, m), 5.98(1H, s), 4.84 (1H, dd, J=13.1, 7.5 Hz), 4.59 (1H, dt, J=8.3, 4.5 Hz),4.19-4.13 (1H, m), 3.79 (6H, s), 3.78-3.72 (1H, m), 3.63-3.40 (3H, m),3.55 (3H, s), 3.36-3.24 (1H, m), 3.09-2.95 (1H, m), 2.59 (1H, septet,J=6.9 Hz), 1.85-1.53 (5H, m), 1.48-1.37 (1H, m), 1.24-1.17 (6H, m), 0.59(3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 155.2 (1P, s).

Example Z-32 Oxazaphospholidine Monomer 22b

Compound 22b was obtained by using 3b instead of 3a in a similar mannerto compound 22a. ¹H NMR (300 MHz, CDCl₃) δ 8.62 (1H, d, J=7.5 Hz),7.57-7.23 (19H, m), 7.02 (1H, d, J=7.5 Hz), 6.89-6.81 (4H, m), 5.92 (1H,s), 4.90 (1H, dt, J=9.0, 5.7 Hz), 4.61 (1H, dt, J=8.7, 4.8 Hz),4.25-4.17 (1H, m), 3.81 (6H, s), 3.67 (1H, d, J=4.5 Hz), 3.62-3.25 (4H,m), 3.38 (3H, s), 3.16-3.02 (1H, m), 2.58 (1H, septet, J=6.9 Hz),1.87-1.40 (6H, m), 1.26-1.14 (6H, m), 0.64 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) δ 158.2 (1P, s).

Example Z-33 Oxazaphospholidine Monomer 23a

Compound 23a was obtained by using“5′-O-(DMTr)-2′-O-methyl-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”instead of “5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 12a. ¹H NMR (300 MHz, CDCl₃) δ 8.67 (1H,brs), 8.01 (1H, s), 7.56-7.16 (24H, m), 6.83-6.74 (4H, m), 6.08 (1H, d,J=6.9 Hz), 4.85-4.76 (1H, m), 4.84 (2H, t, J=6.6 Hz), 4.65-4.56 (1H, m),4.59 (2H, brs), 4.48 (1H, dd, J=6.6, 5.1 Hz), 4.09-4.05 (1H, m), 3.75(6H, s), 3.60-3.42 (2H, m), 3.40-3.26 (2H, m), 3.35 (3H, s), 3.18-3.05(1H, m), 3.08 (2H, t, J=6.6 Hz), 1.89-1.49 (3H, m), 1.48-1.16 (3H, m),0.59 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 156.9 (1P, s).

Example Z-34 Oxazaphospholidine Monomer 23b

Compound 23b was obtained by using 3b instead of 3a in a similar mannerto compound 23a. ¹H NMR (300 MHz, CDCl₃) δ 8.74 (1H, brs), 8.09 (1H, s),7.56-6.94 (24H, m), 6.84-6.71 (4H, m), 6.09 (1H, d, J=4.8 Hz), 4.83-4.70(2H, m), 4.83 (2H, t, J=6.6 Hz), 4.63 (2H, brs), 4.35 (1H, t, J=5.0 Hz),4.23-4.16 (1H, m), 3.75 (6H, s), 3.58-3.19 (4H, m), 3.32 (3H, s),3.16-3.04 (1H, m), 3.07 (2H, t, J=6.6 Hz), 1.90-1.55 (3H, m), 1.48-1.15(3H, m), 0.64 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 154.6 (1P, s).

Example Z-35 Oxazaphospholidine Monomer 24a

Compound 24a was obtained by using“5′-O-(DMTr)-2′-deoxy-2′-fluoro-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”instead of “5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 12a. ¹H NMR (300 MHz, CDCl₃) δ 8.74 (1H,brs), 8.03 (1H, s), 7.55-6.94 (24H, m), 6.80-6.69 (4H, m), 6.21 (1H, dd,J=14.9, 3.6 Hz), 5.34 (1H, dt, J=52.3, 3.6 Hz), 5.01-4.75 (2H, m), 4.84(1H, t, J=6.6 Hz), 4.62 (2H, brs), 4.15-4.07 (1H, m), 3.73 (6H, s),3.59-3.29 (4H, m), 3.15-3.00 (1H, m), 3.07 (2H, t, J=6.6 Hz), 1.90-1.49(3H, m), 1.47-1.12 (3H, m), 0.58 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ155.6 (1P, d, J=10.9 Hz).

Example Z-36 Oxazaphospholidine Monomer 24b

Compound 24b was obtained by using 3b instead of 3a in a similar mannerto compound 24a. ¹H NMR (300 MHz, CDCl₃) δ 8.81 (1H, brs), 8.06 (1H, s),7.55-6.95 (24H, m), 6.77-6.69 (4H, m), 6.06 (1H, d, J=17.1 Hz),5.24-5.08 (1H, m), 5.04-4.80 (2H, m), 4.87 (1H, t, J=6.6 Hz), 4.62 (2H,brs), 4.25-4.19 (1H, m), 3.73 (6H, s), 3.58-3.02 (5H, m), 3.10 (2H, t,J=6.6 Hz), 1.90-1.56 (3H, m), 1.50-1.15 (3H, m), 0.63 (3H, s); ³¹P NMR(121.5 MHz, CDCl₃) δ 158.0 (1P, d, J=4.4 Hz).

Example Z-37 Oxazaphospholidine Monomer 25a

Compound 25a was obtained by using“5′-O-(DMTr)-2′-O-TOM-4-N-(acetyl)cytidine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a. ¹H NMR (300 MHz, CDCl₃) δ 10.04 (1H, brs), 8.30(1H, d, J=7.5 Hz), 7.51-7.21 (19H, m), 6.99 (1H, d, J=7.5 Hz), 6.89-6.81(4H, m), 6.12 (1H, d, J=3.3 Hz), 5.07 (1H, d, J=4.8 Hz), 5.05 (1H, d,J=4.8 Hz), 4.84-4.75 (1H, m), 4.62-4.52 (1H, m), 4.31-4.25 (1H, m),4.08-4.01 (1H, m), 3.78 (6H, d, J=3.0 Hz), 3.55-3.23 (4H, m), 3.10-2.96(1H, m), 2.24 (3H, s), 1.84-1.49 (3H, m), 1.46-0.96 (24H, m), 0.58 (3H,s); ³¹P NMR (121.5 MHz, CDCl₃) δ 156.5 (1P, s).

Example Z-38 Oxazaphospholidine Monomer 25b

Compound 25b was obtained by using 3b instead of 3a in a similar mannerto compound 25a. ¹H NMR (300 MHz, CDCl₃) δ 10.19 (1H, brs), 8.46 (1H, d,J=7.5 Hz), 7.54-7.23 (19H, m), 7.01 (1H, d, J=7.5 Hz), 6.88-6.79 (4H,m), 6.19 (1H, d, J=1.8 Hz), 5.11 (1H, d, J=4.8 Hz), 5.07 (1H, d, J=4.8Hz), 4.81-4.71 (1H, m), 4.60-4.51 (1H, m), 4.26-4.18 (2H, m), 3.79 (6H,s), 3.63-3.55 (1H, m), 3.48-3.28 (2H, m), 3.21-2.94 (2H, m), 2.26 (3H,s), 1.81-1.49 (3H, m), 1.43-0.96 (24H, m), 0.62 (3H, s); ³¹P NMR (121.5MHz, CDCl₃) δ 156.4 (1P, s).

Example Z-39 Oxazaphospholidine Monomer 26a

Compound 26a was obtained by using“5′-O-(DMTr)-2′-deoxy-2′-fluoro-4-N-(isobutyryl)cytidine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a. ¹H NMR (300 MHz, CDCl₃) δ 8.66 (1H, brs), 8.41(1H, d, J=7.5 Hz), 7.55-7.20 (19H, m), 7.01 (1H, d, J=7.5 Hz), 6.89-6.81(4H, m), 6.06 (1H, d, J=15.9 Hz), 4.85 (1H, dd, J=51.4, 3.9 Hz), 4.84(1H, dd, J=12.9, 7.5 Hz), 4.77-4.59 (1H, m), 4.15-4.08 (1H, m), 3.79(6H, s), 3.63-3.29 (4H, m), 3.10-2.96 (1H, m), 2.65 (1H, septet, J=6.9Hz), 1.85-1.53 (3H, m), 1.48-1.17 (3H, m), 1.21 (3H, d, J=4.8 Hz), 1.19(3H, d, J=4.8 Hz), 0.59 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 155.5 (1P,d, J=6.6 Hz).

Example Z-40 Oxazaphospholidine Monomer 26b

Compound 26b was obtained by using 3b instead of 3a in a similar mannerto compound 26a. ¹H NMR (300 MHz, CDCl₃) δ 8.53 (1H, d, J=7.5 Hz),7.57-7.23 (20H, m), 7.10 (11H, d, J=7.5 Hz), 6.89-6.81 (4H, m), 6.10(1H, d, J=15.9 Hz), 5.00-4.92 (1H, m), 4.84 (1H, dd, J=51.5, 3.3 Hz),4.75-4.58 (1H, m), 4.24 (1H, d, J=9.3 Hz), 3.81 (6H, s), 3.65-3.39 (3H,m), 3.32-3.06 (2H, m), 2.59 (1H, septet, J=6.9 Hz), 1.88-1.53 (4H, m),1.49-1.34 (2H, m), 1.27-1.18 (6H, m), 0.65 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) δ 159.0 (1P, d, J=4.4).

Oxazaphospholidine Monomer 27a

Compound 27a was obtained by using“5′-O-(DMTr)-2′-O-methyl-6-N-(benzoyl)adenosine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a.

¹H NMR (300 MHz, CDCl₃) δ 8.66 (1H, s), 8.13 (1H, s), 8.03 (2H, d, J=7.2Hz), 7.64-7.16 (23H, m), 6.79 (4H, d, J=8.7 Hz), 6.08 (1H, d, J=6.3 Hz),4.91-4.81 (1H, m), 4.77-4.69 (1H, m), 4.64-4.57 (1H, m), 4.15-4.10 (1H,m), 3.76 (6H, s), 3.60-3.23 (4H, m), 3.35 (3H, s), 3.14-3.00 (1H, m),1.90-1.19 (6H, m), 0.62 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 155.8 (1P,s).

Oxazaphospholidine Monomer 27b

Compound 27b was obtained by using 3b instead of 3a in a similar mannerto compound 27a.

¹H NMR (300 MHz, CDCl₃) δ 9.12 (1H, brs), 8.73 (1H, s), 8.24 (1H, s),8.07-8.01 (2H, m), 7.62-7.17 (22H, m), 6.83-6.77 (4H, m), 6.12 (1H, d,J=4.8 Hz), 4.84-4.73 (2H, m), 4.43 (1H, t, J=4.8 Hz), 4.25-4.19 (1H, m),3.77 (6H, s), 3.55-3.20 (4H, m), 3.28 (3H, s), 3.16-3.03 (1H, m),1.90-1.17 (6H, m), 0.65 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 155.0 (1P,s).

Oxazaphospholidine Monomer 28a

Compound 28a was obtained by using“5′-O-(DMTr)-2′-deoxy-2′-fluoro-6-N-(benzoyl)adenosine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a.

¹H NMR (300 MHz, CDCl₃) δ 8.64 (1H, s), 8.14 (1H, s), 8.06-8.01 (2H, m),7.63-7.07 (23H, m), 6.78-6.70 (4H, m), 6.12 (1H, dd, J=18.0, 2.4 Hz),5.24-5.01 (2H, m), 4.94-4.84 (1H, m), 4.17-4.06 (1H, m), 3.73 (6H, s),3.55-3.40 (3H, m), 3.30-3.22 (1H, m), 3.03-2.88 (1H, m), 1.92-1.19 (6H,m), 0.62 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 150.5 (1P, d, J=7.7 Hz).

Oxazaphospholidine Monomer 28b

Compound 28b was obtained by using 3b instead of 3a in a similar mannerto compound 28a.

¹H NMR (300 MHz, CDCl₃) δ 9.07 (1H, brs), 8.80 (1H, s), 8.24 (1H, s),8.08-8.01 (2H, m), 7.66-7.15 (22H, m), 6.81-6.75 (4H, m), 6.14 (1H, dd,J=18.0, 1.8 Hz), 5.16-4.91 (3H, m), 4.28-4.21 (1H, m), 3.76 (6H, s),3.57-3.11 (5H, m), 1.82-1.16 (6H, m), 0.65 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) δ 157.8 (1P, d, J=5.6 Hz).

Oxazaphospholidine Monomer 29a

Compound 29a was obtained by using“5′-O-(DMTr)-2′-O-TOM-2-N-(acetyl)guanosine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a.

¹H NMR (300 MHz, CDCl₃) δ 7.70 (1H, s), 7.63-7.13 (21H, m), 6.84-6.76(4H, m), 5.77 (1H, d, J=8.4 Hz), 5.41-5.33 (1H, m), 4.90 (2H, s),4.78-4.68 (2H, m), 3.86 (1H, brs), 3.75 (3H, s), 3.74 (3H, s), 3.56-3.41(2H, m), 3.32-2.90 (3H, m), 1.92-1.10 (9H, m), 0.97-0.87 (21H, m), 0.52(3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 158.1 (1P, s).

Oxazaphospholidine Monomer 29b

Compound 29b was obtained by using 3b instead of 3a in a similar mannerto compound 29a.

¹H NMR (300 MHz, CDCl₃) δ 7.77 (1H, s), 7.56-7.15 (21H, m), 6.82-6.75(4H, m), 5.86 (1H, d, J=7.5 Hz), 5.26-5.17 (1H, m), 4.95 (1H, d, J=5.4Hz), 4.85 (1H, d, J=5.4 Hz), 4.78-4.71 (1H, m), 4.59-4.49 (1H, m),4.10-4.05 (1H, m), 3.74 (6H, s), 3.52-3.37 (2H, m), 3.30-3.18 (1H, m),3.11-2.85 (2H, m), 1.85-1.15 (9H, m), 0.93-0.84 (21H, m), 0.62 (3H, s);³¹P NMR (121.5 MHz, CDCl₃) δ 152.3 (1P, s).

Oxazaphospholidine Monomer 30a

Compound 30a was obtained by using “5′-O-(DMTr)-2′-O-TOM-uridine”instead of “5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 12a.

¹H NMR (300 MHz, CDCl₃) δ 7.76 (1H, d, J=8.1 Hz), 7.55-7.18 (20H, m),6.88-6.80 (4H, m), 6.11 (1H, d, J=6.0 Hz), 5.32 (1H, d, J=8.1 Hz), 4.99(1H, d, J=5.1 Hz), 4.93 (1H, d, J=5.1 Hz), 4.84-4.75 (1H, m), 4.54-4.46(1H, m), 4.38 (1H, t, J=5.7 Hz), 3.87-3.83 (1H, m), 3.78 (3H, s), 3.77(3H, s), 3.56-3.42 (1H, m), 3.39-3.28 (1H, m), 3.36 (1H, dd, J=11.0, 2.7Hz), 3.25 (1H, dd, J=11.0, 2.7 Hz), 3.16-3.03 (1H, m), 1.88-1.12 (6H,m), 1.08-0.97 (21H, m), 0.59 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 156.6(1P, s).

Oxazaphospholidine Monomer 30b

Compound 30b was obtained by using 3b instead of 3a in a similar mannerto compound 30a.

¹H NMR (600 MHz, CDCl₃) δ 7.87 (1H, d, J=7.8 Hz), 7.52-7.48 (4H, m),7.38-7.21 (16H, m), 6.83-6.79 (4H, m), 6.14 (1H, d, J=4.8 Hz), 5.33 (1H,d, J=7.8 Hz), 4.99 (1H, d, J=5.4 Hz), 4.89 (1H, d, J=5.4 Hz), 4.67 (1H,dd, J=13.8, 7.2 Hz), 4.52 (1H, dt, J=10.4, 4.8 Hz), 4.31 (1H, t, J=4.8Hz), 4.06-4.03 (1H, m), 3.78 (3H, s), 3.77 (3H, s), 3.47 (1H, dd,J=10.4, 2.4 Hz), 3.47-3.39 (1H, m), 3.22-3.17 (2H, m), 3.00 (1H, ddd,J=19.5, 10.4, 4.8 Hz), 1.82-1.74 (1H, m), 1.68-1.58 (1H, m), 1.56 (1H,dd, J=14.4, 8.4 Hz), 1.38 (1H, dd, J=14.4, 7.2 Hz), 1.31-1.25 (1H, m),1.26-1.17 (1H, m), 1.08-0.98 (21H, m), 0.63 (3H, s); ³¹P NMR (243.0 MHz,CDCl₃) δ 154.3 (1P, s).

Oxazaphospholidine Monomer 31a

Compound 31a was obtained by using“5′-O-(DMTr)-2′-O,4′-C-methylene-6-N-(benzoyl)adenosine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a.

¹H NMR (300 MHz, CDCl₃) δ 9.10 (1H, brs), 8.76 (1H, s), 8.32 (1H, s),8.04 (2H, d, J=7.2 Hz), 7.64-7.18 (22H, m), 6.84 (4H, d, J=8.7 Hz), 6.10(1H, s), 4.76 (1H, d J=6.9 Hz), 4.58 (1H, s), 4.61-4.51 (1H, m), 3.91(1H, d, J=7.8 Hz), 3.77 (1H, d, J=7.8 Hz), 3.75 (6H, s), 3.50 (1H, s),3.47-3.33 (1H, m), 3.31-3.19 (1H, m), 3.03-2.88 (1H, m), 1.84-1.09 (6H,m), 0.51 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 152.9 (1P, s).

Oxazaphospholidine Monomer 31b

Compound 31b was obtained by using 3b instead of 3a in a similar mannerto compound 31a.

¹H NMR (300 MHz, CDCl₃) δ 8.81 (1H, s), 8.30 (1H, s), 8.07-8.00 (2H, m),7.64-7.17 (22H, m), 6.86-6.79 (4H, m), 6.12 (1H, s), 4.81-4.72 (1H, m),4.62 (1H, d J=7.2 Hz), 4.57 (1H, s), 3.94 (1H, d, J=7.8 Hz), 3.89 (1H,d, J=7.8 Hz), 3.77 (6H, s), 3.48 (2H, s), 3.46-3.32 (1H, m), 3.24-3.13(1H, m), 3.10-2.97 (1H, m), 1.84-1.49 (3H, m), 1.42-1.09 (3H, m), 0.58(3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 157.3 (1P, s).

Oxazaphospholidine Monomer 32a

Compound 32a was obtained by using“5′-O-(DMTr)-2′-O,4′-C-methylene-4-N-(isobutyryl)-5-methylcytidine”instead of “5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 12a.

¹H NMR (300 MHz, CDCl₃) δ 7.88 (1H, brs), 7.58-7.18 (20H, m), 6.88-6.80(4H, m), 5.65 (1H, s), 4.69-4.60 (1H, m), 4.52 (1H, d, J=6.6 Hz), 4.49(1H, s), 3.81-3.74 (1H, m), 3.75 (3H, s), 3.73 (3H, s), 3.64 (1H, d,J=8.1 Hz), 3.56 (1H, d, J=11.1 Hz), 3.53 (1H, d, J=8.1 Hz), 3.46 (1H, d,J=11.1 Hz), 3.56-3.40 (1H, m), 3.32-3.20 (1H, m), 3.14-3.00 (1H, m),1.85-1.12 (6H, m), 1.60 (3H, s), 1.19 (6H, d, J=6.9 Hz), 0.55 (3H, s);³¹P NMR (121.5 MHz, CDCl₃) δ 155.9 (1P, s).

Oxazaphospholidine Monomer 32b

Compound 32b was obtained by using 3b instead of 3a in a similar mannerto compound 32a.

¹H NMR (300 MHz, CDCl₃) δ 7.86 (1H, brs), 7.56-7.19 (20H, m), 6.88-6.79(4H, m), 5.69 (1H, s), 4.86-4.76 (1H, m), 4.46 (1H, s), 4.45 (1H, d,J=7.5 Hz), 3.80-3.75 (1H, m), 3.79 (6H, s), 3.74 (1H, d, J=8.1 Hz), 3.69(1H, d, J=8.1 Hz), 3.51 (1H, d, J=11.1 Hz), 3.44-3.30 (1H, m), 3.39 (1H,d, J=11.1 Hz), 3.29-3.17 (1H, m), 3.11-2.97 (1H, m), 1.86-1.52 (3H, m),1.64 (3H, s), 1.45-1.10 (3H, m), 1.21 (6H, d, J=6.6 Hz), 0.62 (3H, s);³¹P NMR (121.5 MHz, CDCl₃) δ 158.2 (1P, s).

Oxazaphospholidine Monomer 33a

Compound 33a was obtained by using“5′-O-(DMTr)-2′-O,4′-C-methylene-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”instead of “5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 12a.

¹H NMR (300 MHz, CDCl₃) δ 8.71 (1H, brs), 8.16 (1H, s), 7.50-7.17 (21H,m), 7.09-7.01 (3H, m), 6.86-6.79 (4H, m), 6.03 (1H, s), 4.84 (2H, t,J=6.6 Hz), 4.72 (2H, s), 4.68 (1H, d, J=7.2 Hz), 4.55-4.46 (1H, m), 4.50(1H, s), 3.90 (1H, d, J=7.8 Hz), 3.77 (1H, d, J=7.8 Hz), 3.75 (6H, s),3.51 (1H, d, J=10.8 Hz), 3.47 (1H, d, J=10.8 Hz), 3.45-3.21 (2H, m),3.08 (2H, t, J=6.6 Hz), 3.03-2.89 (1H, m), 1.80-1.08 (6H, m), 0.47 (3H,s); ³¹P NMR (121.5 MHz, CDCl₃) δ 153.2 (1P, s).

Oxazaphospholidine Monomer 33b

Compound 33b was obtained by using 3b instead of 3a in a similar mannerto compound 33a.

¹H NMR (300 MHz, CDCl₃) δ 8.86 (1H, brs), 8.13 (1H, s), 7.55-7.17 (21H,m), 7.08-6.98 (3H, m), 6.95-6.78 (4H, m), 6.01 (1H, s), 4.86 (2H, t,J=6.6 Hz), 4.82-4.73 (1H, m), 4.70 (2H, s), 4.64 (1H, d, J=7.5 Hz), 4.49(1H, s), 3.94 (1H, d, J=7.8 Hz), 3.89 (1H, d, J=7.8 Hz), 3.77 (6H, s),3.46 (2H, s), 3.45-3.30 (1H, m), 3.24-3.12 (1H, m), 3.09 (2H, t, J=6.6Hz), 3.09-2.96 (1H, m), 1.81-1.50 (3H, m), 1.41-1.06 (3H, m), 0.58 (3H,s); ³¹P NMR (121.5 MHz, CDCl₃) δ 157.4 (1P, s).

Oxazaphospholidine Monomer 34a

Compound 34a was obtained by using“5′-O-(DMTr)-2′-O,4′-C-methylene-5-methyluridine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 12a.

¹H NMR (300 MHz, CDCl₃) δ 7.71 (1H, d, J=0.9 Hz), 7.50-7.17 (20H, m),6.87-6.80 (4H, m), 5.61 (1H, s), 4.69-4.60 (1H, m), 4.55 (1H, d, J=6.9Hz), 4.41 (1H, s), 3.74 (3H, s), 3.73 (3H, s), 3.64 (1H, d, J=7.8 Hz),3.55 (1H, d, J=7.8 Hz), 3.53 (1H, d, J=10.8 Hz), 3.46 (1H, d, J=10.8Hz), 3.56-3.42 (1H, m), 3.35-3.24 (1H, m), 3.13-3.00 (1H, m), 1.85-1.45(3H, m), 1.55 (3H, d, J=0.9 Hz), 1.41-1.12 (3H, m), 0.56 (3H, s); ³¹PNMR (121.5 MHz, CDCl₃) δ 155.1 (1P, s).

Oxazaphospholidine Monomer 34b

Compound 34b was obtained by using 3b instead of 3a in a similar mannerto compound 34a.

¹H NMR (300 MHz, CDCl₃) δ 7.69 (1H, s), 7.56-7.19 (20H, m), 6.88-6.79(4H, m), 5.66 (1H, s), 4.87-4.77 (1H, m), 4.47 (1H, d, J=7.8 Hz), 4.40(1H, s), 3.78 (6H, s), 3.74 (1H, d, J=7.8 Hz), 3.68 (1H, d, J=7.8 Hz),3.50 (1H, d, J=10.8 Hz), 3.46-3.32 (1H, m), 3.39 (1H, d, J=10.8 Hz),3.30-3.19 (1H, m), 3.12-2.98 (1H, m), 1.85-1.56 (3H, m), 1.59 (31, s),1.46-1.12 (3H, m), 0.63 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) δ 158.1 (1P,s).

Oxazaphospholidine Monomer 35a

Compound 35a was obtained by using 13a′ instead of 3a in a similarmanner to compound 13a. ¹H NMR (600 MHz, CDCl₃) δ 7.76 (2H, d, J=9.0Hz), 7.62 (1H, d, J=1.2 Hz), 7.40 (2H, d, J=7.2 Hz), 7.32-7.23 (101, m),6.85 (4H, d, J=8.4 Hz), 6.41 (1H, dd, J=8.4, 5.4 Hz), 4.94 (1H, dd,J=12.3, 5.4 Hz), 4.84-4.79 (1H, m), 4.03-4.01 (1H, m), 3.79 (6H, s),3.59-3.53 (1H, m), 3.52-3.44 (2H, m), 3.41 (1H, dd, J=14.7, 7.2 Hz),3.37-3.30 (2H, m), 3.13 (1H, ddd, J=19.3, 10.3, 4.1 Hz), 2.50-2.44 (1H,m), 2.39 (3H, s), 2.35-2.29 (1H, m), 1.91-1.72 (2H, m), 1.64-1.59 (1H,m), 1.40 (3H, s), 1.12-1.05 (1H, m); ³¹P NMR (243.0 MHz, CDCl₃) δ 154.2(1P, s).

Example Z-41

The above Compound Z-27, which represents a conventional monomer, wasused to produce oligos. FIG. 70 shows a chart of products obtainedthrough Comparison Example Z-1. As shown in FIGS. 69 and 70, the presentmonomers provided more complete deprotection and less side product,which makes product isolation and/or purification easier.

In some embodiments, the present invention provides chemically stablemonomers. Exemplary such monomers are depicted in the Examples above. Insome embodiments, the present invention provides monomers with highisolated yield. In some embodiments, the present invention providesmonomers with isolated yield higher than conventional method. In someembodiments, the isolated yield is more than 80%. Exemplary suchmonomers are depicted in the Examples above.

Condensing Reagent

Condensing reagents (C_(R)) useful in accordance with methods of thepresent invention are of any one of the following general formulae:

wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷, Z⁸, and Z⁹ are independentlyoptionally substituted group selected from alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, or heteroaryloxy, or wherein any of Z²and Z³, Z⁵ and Z⁶, Z⁷ and Z⁸, Z⁸ and Z⁹, Z⁹ and Z⁷, or Z⁷ and Z⁸ and Z⁹are taken together to form a 3 to 20 membered alicyclic or heterocyclicring; Q⁻ is a counter anion; and LG is a leaving group.

In some embodiments, a counter ion of a condensing reagent C_(R) is Cl⁻,Br⁻, BF₄ ⁻, PF₆ ⁻, TfO⁻, Tf₂N⁻, AsF₆ ⁻, ClO₄ ⁻, or SbF₆ ⁻, wherein Tf isCF₃SO₂. In some embodiments, a leaving group of a condensing reagentC_(R) is F, Cl, Br, I, 3-nitro-1,2,4-triazole, imidazole, alkyltriazole,tetrazole, pentafluorobenzene, or 1-hydroxybenzotriazole.

Examples of condensing reagents used in accordance with methods of thepresent invention include, but are not limited to, pentafluorobenzoylchloride, carbonyldiimidazole (CDI),1-mesitylenesulfonyl-3-nitrotriazole (MSNT),1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDCI-HCl),benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(PyBOP), N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BopCl),2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), andO-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU),DIPCDI; N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic bromide (BopBr),1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP),3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP), bromotripyrrolidinophosphoniumhexafluorophosphate (PyBrOP);O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU); and tetramethylfluoroformamidinium hexafluorophosphate (TFFH).In certain embodiments, a counter ion of the condensing reagent C_(R) isCl⁻, Br⁻, BF₄ ⁻, PF₆ ⁻, TfO⁻, Tf₂N⁻, AsF₆ ⁻, ClO₄ ⁻, or SbF₆ ⁻, whereinTf is CF₃SO₂.

In some embodiments, a condensing reagent is1-(2,4,6-triisopropylbenzenesulfonyl)-5-(pyridin-2-yl) tetrazolide,pivaloyl chloride, bromotrispyrrolidinophosphonium hexafluorophosphate,N,N′-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BopCl), or2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane. In some embodiment,a condensing reagent is N,N′-bis(2-oxo-3-oxazolidinyl)phosphinicchloride (BopCl). In some embodiments, a condensing reagent is selectedfrom those described in WO/2006/066260).

In some embodiments, a condensing reagent is1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP), or3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP):

Selection of Base and Sugar of Nucleoside Coupling Partner

As described herein, nucleoside coupling partners for use in accordancewith methods of the present invention can be the same as one another orcan be different from one another. In some embodiments, nucleosidecoupling partners for use in the synthesis of a provided oligonucleotideare of the same structure and/or stereochemical configuration as oneanother. In some embodiments, each nucleoside coupling partner for usein the synthesis of a provided oligonucleotide is not of the samestructure and/or stereochemical configuration as certain othernucleoside coupling partners of the oligonucleotide. Exemplarynucleobases and sugars for use in accordance with methods of the presentinvention are described herein. One of skill in the relevant chemicaland synthetic arts will recognize that any combination of nucleobasesand sugars described herein are contemplated for use in accordance withmethods of the present invention.

Coupling Step:

Exemplary coupling procedures and chiral reagents and condensingreagents for use in accordance with the present invention are outlinedin, inter alia, Wada I (JP4348077; WO2005/014609; WO2005/092909), WadaII (WO2010/064146), and Wada III (WO2012/039448). Chiral nucleosidecoupling partners for use in accordance with the present invention arealso referred to herein as “Wada amidites.” In some embodiments, acoupling partner has the structure of

wherein B^(PRO) is a protected nucleobase. In some embodiments, acoupling partner has the structure of

wherein B^(PRO) is a protected nucleobase. Exemplary chiralphosphoramidites as coupling partner are depicted below:

One of the methods used for synthesizing the coupling partner isdepicted in Scheme II below.

In some embodiments, the step of coupling comprises reacting a freehydroxyl group of a nucleotide unit of an oligonucleotide with anucleoside coupling partner under suitable conditions to effect thecoupling. In some embodiments, the step of coupling is preceded by astep of deblocking. For instance, in some embodiments, the 5′ hydroxylgroup of the growing oligonucleotide is blocked (i.e., protected) andmust be deblocked in order to subsequently react with a nucleosidecoupling partner.

Once the appropriate hydroxyl group of the growing oligonucleotide hasbeen deblocked, the support is washed and dried in preparation fordelivery of a solution comprising a chiral reagent and a solutioncomprising an activator. In some embodiments, a chiral reagent and anactivator are delivered simultaneously. In some embodiments, co-deliverycomprises delivering an amount of a chiral reagent in solution (e.g., aphosphoramidite solution) and an amount of activator in a solution(e.g., a CMPT solution) in a polar aprotic solvent such as a nitrilesolvent (e.g., acetonitrile).

In some embodiments, the step of coupling provides a crude productcomposition in which the chiral phosphite product is present in adiastereomeric excess of >95%. In some embodiments, the chiral phosphiteproduct is present in a diastereomeric excess of >96%. In someembodiments, the chiral phosphite product is present in a diastereomericexcess of >97%. In some embodiments, the chiral phosphite product ispresent in a diastereomeric excess of >98%. In some embodiments, thechiral phosphite product is present in a diastereomeric excess of >99%.

Capping Step:

Provided methods for making chirally controlled oligonucleotidescomprise a step of capping. In some embodiments, a step of capping is asingle step. In some embodiments, a step of capping is two steps. Insome embodiments, a step of capping is more than two steps.

In some embodiments, a step of capping comprises steps of capping thefree amine of the chiral auxiliary and capping any residual unreacted 5′hydroxyl groups. In some embodiments, the free amine of the chiralauxiliary and the unreacted 5′ hydroxyl groups are capped with the samecapping group. In some embodiments, the free amine of the chiralauxiliary and the unreacted 5′ hydroxyl groups are capped with differentcapping groups. In certain embodiments, capping with different cappinggroups allows for selective removal of one capping group over the otherduring synthesis of the oligonucleotide. In some embodiments, thecapping of both groups occurs simultaneously. In some embodiments, thecapping of both groups occurs iteratively.

In certain embodiments, capping occurs iteratively and comprises a firststep of capping the free amine followed by a second step of capping thefree 5′ hydroxyl group, wherein both the free amine and the 5′ hydroxylgroup are capped with the same capping group. For instance, in someembodiments, the free amine of the chiral auxiliary is capped using ananhydride (e.g., phenoxyacetic anhydride, i.e., Pac₂O) prior to cappingof the 5′ hydroxyl group with the same anhydride. In certainembodiments, the capping of the 5′ hydroxyl group with the sameanhydride occurs under different conditions (e.g., in the presence ofone or more additional reagents). In some embodiments, capping of the 5′hydroxyl group occurs in the presence of an amine base in an etherialsolvent (e.g., NMI (N-methylimidazole) in THF). The phrase “cappinggroup” is used interchangeably herein with the phrases “protectinggroup” and “blocking group”.

In some embodiments, an amine capping group is characterized in that iteffectively caps the amine such that it prevents rearrangement and/ordecomposition of the intermediate phosphite species. In someembodiments, a capping group is selected for its ability to protect theamine of the chiral auxiliary in order to prevent intramolecularcleavage of the internucleotide linkage phosphorus.

In some embodiments, a 5′ hydroxyl group capping group is characterizedin that it effectively caps the hydroxyl group such that it prevents theoccurrence of “shortmers,” e.g., “n-m” (m and n are integers and m<n; nis the number of bases in the targeted oligonucleotide) impurities thatoccur from the reaction of an oligonucleotide chain that fails to reactin a first cycle but then reacts in one or more subsequent cycles. Thepresence of such shortmers, especially “n−1”, has a deleterious effectupon the purity of the crude oligonucleotide and makes finalpurification of the oligonucleotide tedious and generally low-yielding.

In some embodiments, a particular cap is selected based on its tendencyto facilitate a particular type of reaction under particular conditions.For instance, in some embodiments, a capping group is selected for itsability to facilitate an E1 elimination reaction, which reaction cleavesthe cap and/or auxiliary from the growing oligonucleotide. In someembodiments, a capping group is selected for its ability to facilitatean E2 elimination reaction, which reaction cleaves the cap and/orauxiliary from the growing oligonucleotide. In some embodiments, acapping group is selected for its ability to facilitate a β-eliminationreaction, which reaction cleaves the cap and/or auxiliary from thegrowing oligonucleotide.

Modifying Step:

As used herein, the phrase “modifying step”, “modification step” and“P-modification step” are used interchangeably and refer generally toany one or more steps used to install a modified internucleotidiclinkage. In some embodiments, the modified internucleotidic linkagehaving the structure of formula I. A P-modification step of the presentinvention occurs during assembly of a provided oligonucleotide ratherthan after assembly of a provided oligonucleotide is complete. Thus,each nucleotide unit of a provided oligonucleotide can be individuallymodified at the linkage phosphorus during the cycle within which thenucleotide unit is installed.

In some embodiments, a suitable P-modification reagent is a sulfurelectrophile, selenium electrophile, oxygen electrophile, boronatingreagent, or an azide reagent.

For instance, in some embodiments, a selemium reagent is elementalselenium, a selenium salt, or a substituted diselenide. In someembodiments, an oxygen electrophile is elemental oxygen, peroxide, or asubstituted peroxide. In some embodiments, a boronating reagent is aborane-amine (e.g., N,N-diisopropylethylamine (BH₃.DIPEA),borane-pyridine (BH₃.Py), borane-2-chloropyridine (BH₃.CPy),borane-aniline (BH₃.An)), a borane-ether reagent (e.g.,borane-tetrahydrofuran (BH₃.THF)), a borane-dialkylsulfide reagent(e.g., BH₃.Me₂S), aniline-cyanoborane, or atriphenylphosphine-carboalkoxyborane. In some embodiments, an azidereagent is comprises an azide group capable of undergoing subsequentreduction to provide an amine group.

In some embodiments, a P-modification reagent is a sulfurization reagentas described herein. In some embodiments, a step of modifying comprisessulfurization of phosphorus to provide a phosphorothioate linkage orphosphorothioate triester linkage. In some embodiments, a step ofmodifying provides an oligonucleotide having an internucleotidic linkageof formula I.

In some embodiments, the present invention provides sulfurizingreagents, and methods of making, and use of the same.

In some embodiments, such sulfurizing reagents are thiosulfonatereagents. In some embodiments, a thiosulfonate reagent has a structureof formula S-I:

wherein:

R^(s1) is R; and

each of R, L and R¹ is independently as defined and described above andherein.

In some embodiments, the sulfurizing reagent is a bis(thiosulfonate)reagent. In some embodiments, the bis(thiosulfonate) reagent has thestructure of formula S-II:

wherein each of R^(s1) and L is independently as defined and describedabove and herein.

As defined generally above, R^(s1) is R, wherein R is as defined anddescribed above and herein. In some embodiments, R^(s1) is optionallysubstituted aliphatic, aryl, heterocyclyl or heteroaryl. In someembodiments, R^(s1) is optionally substituted alkyl. In someembodiments, R^(s1) is optionally substituted alkyl. In someembodiments, R^(s1) is methyl. In some embodiments, R^(s1) iscyanomethyl. In some embodiments, R^(s1) is nitromethyl. In someembodiments, R^(s1) is optionally substituted aryl. In some embodiments,R^(s1) is optionally substituted phenyl. In some embodiments, R^(s1) isphenyl. In some embodiments, R^(s1) is p-nitrophenyl. In someembodiments, R^(s1) is p-methylphenyl. In some embodiments, R^(s1) isp-chlorophenyl. In some embodiments, R^(s1) is o-chlorophenyl. In someembodiments, R^(s1) is 2,4,6-trichlorophenyl. In some embodiments,R^(s1) is pentafluorophenyl. In some embodiments, R^(s1) is optionallysubstituted heterocyclyl. In some embodiments, R^(s1) is optionallysubstituted heteroaryl.

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is —S—R^(L3)— or —S—C(O)—R^(L3)—. In some embodiments, Lis —S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3) is an optionallysubstituted C₁-C₆ alkylene. In some embodiments, L is —S—R^(L3)— or—S—C(O)—R^(L3)—, wherein R^(L3) is an optionally substituted C₁-C₆alkenylene. In some embodiments, L is —S—R^(L3)— or —S—C(O)—R^(L3)—,wherein R^(L3) is an optionally substituted C₁-C₆ alkylene wherein oneor more methylene units are optionally and independently replaced by anoptionally substituted C₁-C₆ alkenylene, arylene, or heteroarylene. Insome embodiments, In some embodiments, R^(L3) is an optionallysubstituted —S—(C₁-C₆ alkenylene)-, —S—(C₁-C₆ alkylene)-, —S—(C₁-C₆alkylene)-arylene-(C₁-C₆ alkylene)-, —S—CO-arylene-(C₁-C₆ alkylene)-, or—S—CO—(C₁-C₆ alkylene)-arylene-(C₁-C₆ alkylene)-. In some embodiments,the sulfurizing reagent has the structure of S-I or S-II, wherein L is—S—R^(L3)— or —S—C(O)—R^(L3)—, and the sulfur atom is connected to R¹.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is alkylene, alkenylene, arylene or heteroarylene.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is

In some embodiments, L is

wherein the sulfur atom is connected to R¹.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein R¹ is

In some embodiments, R¹ is

wherein the sulfur atom is connected to L.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is

wherein the sulfur atom is connected to R¹; and R¹ is

wherein the sulfur atom is connected to L.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein R¹ is —S—R^(L2), wherein R^(L2) is as defined anddescribed above and herein. In some embodiments, R^(L2) is an optionallysubstituted group selected from —S—(C₁-C₆ alkylene)-heterocyclyl,—S—(C₁-C₆ alkenylene)-heterocyclyl, —S—(C₁-C₆ alkylene)-N(R′)₂,—S—(C₁-C₆ alkylene)-N(R′)₃, wherein each R′ is as defined above anddescribed herein.

In some embodiments, -L-R¹ is —R^(L3)—S—R^(L2), wherein each variable isindependently as defined above and described herein. In someembodiments, -L-R¹ is —R^(L3)—C(O)—S—S—R^(L2), wherein each variable isindependently as defined above and described herein.

Exemplary bis(thiosulfonate) reagents of formula S-II are depictedbelow:

In some embodiments, the sulfurization reagent is a compound having oneof the following formulae:

S₈,R^(s2)—S—S—R^(s3), or R^(s2)—S—X^(s)—R^(s3),

wherein:

-   each of R^(s2) and R^(s3) is independently an optionally substituted    group selected from aliphatic, aminoalkyl, carbocyclyl,    heterocyclyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy,    heteroaryloxy, acyl, amide, imide, or thiocarbonyl; or    -   R^(s2) and R^(s3) are taken together with the atoms to which        they are bound to form an optionally substituted heterocyclic or        heteroaryl ring;-   X^(s) is —S(O)₂—, —O—, or —N(R′)—; and-   R′ is as defined and described above and herein.

In some embodiments, the sulfurization reagent is S₈,

In some embodiments, the sulfurization reagent is S₈,

In some embodiments, the sulfurization reagent is

Exemplary sulfuring reagents are depicted in Table 5 below.

TABLE 5 Exemplary sulfurization reagents.

S₈

In some embodiments, a provided sulfurization reagent is used to modifyan H-phosphonate. For instance, in some embodiments, an H-phosphonateoligonucleotide is synthesized using, e.g., a method of Wada I or WadaII, and is modified using a sulfurization reagent of formula S-I orS-II:

wherein each of R^(S1), L, and R¹ are as described and defined above andherein.

In some embodiments, the present invention provides a process forsynthesizing a phosphorothioate triester, comprising steps of:

i) reacting an H-phosphonate of structure:

wherein each of W, Y, and Z are as described and defined above andherein, with a silylating reagent to provide a silyloxyphosphonate; and

ii) reacting the silyloxyphosphonate with a sulfurization reagent ofstructure S-I or S-II:

to provide a phosphorothiotriester.

In some embodiments, a selenium electrophile is used instead of asulfurizing reagent to introduce modification to the internucleotidiclinkage. In some embodiments, a selenium electrophile is a compoundhaving one of the following formulae:

Se, R^(s2)—Se—Se—R^(s3), or R^(s2)—Se—X^(s)—R^(s3),

wherein:

-   each of R^(s2) and R^(s3) is independently an optionally substituted    group selected from aliphatic, aminoalkyl, carbocyclyl,    heterocyclyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy,    heteroaryloxy, acyl, amide, imide, or thiocarbonyl; or    -   R^(s2) and R^(s3) are taken together with the atoms to which        they are bound to form an optionally substituted heterocyclic or        heteroaryl ring;-   X^(s) is —S(O)₂—, —O—, or —N(R′)—; and-   R′ is as defined and described above and herein.

In other embodiments, the selenium electrophile is a compound of Se,KSeCN,

In some embodiments, the selenium electrophile is Se or

In some embodiments, a sulfurization reagent for use in accordance withthe present invention is characterized in that the moiety transferred tophosphorus during sulfurization is a substituted sulfur (e.g., —SR) asopposed to a single sulfur atom (e.g., —S⁻ or ═S).

In some embodiments, a sulfurization reagent for use in accordance withthe present invention is characterized in that the activity of thereagent is tunable by modifying the reagent with a certain electronwithdrawing or donating group.

In some embodiments, a sulfurization reagent for use in accordance withthe present invention is characterized in that it is crystalline. Insome embodiments, a sulfurization reagent for use in accordance with thepresent invention is characterized in that it has a high degree ofcrystallinity. In certain embodiments, a sulfurization reagent for usein accordance with the present invention is characterized by ease ofpurification of the reagent via, e.g., recrystallization. In certainembodiments, a sulfurization reagent for use in accordance with thepresent invention is characterized in that it is substantially free fromsulfur-containing impurities. In some embodiments, sulfurizationreagents which are substantially free from sulfur-containing impuritiesshow increased efficiency.

In some embodiments, the provided chirally controlled oligonucleotidecomprises one or more phosphate diester linkages. To synthesize suchchirally controlled oligonucleotides, one or more modifying steps areoptionally replaced with an oxidation step to install the correspondingphosphate diester linkages. In some embodiments, the oxidation step isperformed in a fashion similar to ordinary oligonucleotide synthesis. Insome embodiments, an oxidation step comprises the use of I₂. In someembodiments, an oxidation step comprises the use of I₂ and pyridine. Insome embodiments, an oxidation step comprises the use of 0.02 M I₂ in aTHF/pyridine/water (70:20:10—v/v/v) co-solvent system. An exemplarycycle is depicted in Scheme I-c.

In some embodiments, a phosphorothioate precursor is used to synthesizechirally controlled oligonucleotides comprising phosphorothioatelinkages. In some embodiments, such a phosphorothioate precursor is

In some embodiments,

is converted into phosphorothioate diester linkages during standarddeprotection/release procedure after cycle exit. Examples are furtherdepicted below.

In some embodiments, the provided chirally controlled oligonucleotidecomprises one or more phosphate diester linkages and one or morephosphorothioate diester linkages. In some embodiments, the providedchirally controlled oligonucleotide comprises one or more phosphatediester linkages and one or more phosphorothioate diester linkages,wherein at least one phosphate diester linkage is installed after allthe phosphorothioate diester linkages when synthesized from 3′ to 5′. Tosynthesize such chirally controlled oligonucleotides, in someembodiments, one or more modifying steps are optionally replaced with anoxidation step to install the corresponding phosphate diester linkages,and a phosphorothioate precursor is installed for each of thephosphorothioate diester linkages. In some embodiments, aphosphorothioate precursor is converted to a phosphorothioate diesterlinkage after the desired oligonucleotide length is achieved. In someembodiments, the deprotection/release step during or after cycle exitconverts the phosphorothioate precursors into phosphorothioate diesterlinkages. In some embodiments, a phosphorothioate precursor ischaracterized in that it has the ability to be removed by abeta-elimination pathway. In some embodiments, a phosphorothioateprecursor is

As understood by one of ordinary skill in the art, one of the benefitsof using a phosphorothioate precursor, for instance,

during synthesis is that

is more stable than phosphorothioate in certain conditions.

In some embodiments, a phosphorothioate precursor is a phosphorusprotecting group as described herein, e.g., 2-cyanoethyl (CE or Cne),2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl,o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl,3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl,2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl,2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl,4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl. Examples are furtherdepicted below.

Methods for synthesizing a desired sulfurization reagent are describedherein and in the examples section.

As noted above, in some embodiments, sulfurization occurs underconditions which cleave the chiral reagent from the growingoligonucleotide. In some embodiments, sulfurization occurs underconditions which do not cleave the chiral reagent from the growingoligonucleotide.

In some embodiments, a sulfurization reagent is dissolved in a suitablesolvent and delivered to the column. In certain embodiments, the solventis a polar aprotic solvent such as a nitrile solvent. In someembodiments, the solvent is acetonitrile. In some embodiments, asolution of sulfurization reagent is prepared by mixing a sulfurizationreagent (e.g., a thiosulfonate derivative as described herein) withBSTFA (N,O-bis-trimethylsilyl-trifluoroacetamide) in a nitrile solvent(e.g., acetonitrile). In some embodiments, BSTFA is not included. Forexample, the present inventors have found that relatively more reactivesulfurization reagents of general formula R^(s2)—S—S(O)₂—R^(s3) canoften successfully participate in sulfurization reactions in the absenceof BSTFA. To give but one example, the inventors have demonstrated thatwhere R^(s2) is p-nitrophenyl and R^(s3) is methyl then no BSTFA isrequired. In light of this disclosure, those skilled in the art willreadily be able to determine other situations and/or sulfurizationreagents that do not require BSTFA.

In some embodiments, the sulfurization step is performed at roomtemperature. In some embodiments, the sulfurization step is performed atlower temperatures such as about 0° C., about 5° C., about 10° C., orabout 15° C. In some embodiments, the sulfurization step is performed atelevated temperatures of greater than about 20° C.

In some embodiments, a sulfurization reaction is run for about 1 minuteto about 120 minutes. In some embodiments, a sulfurization reaction isrun for about 1 minute to about 90 minutes. In some embodiments, asulfurization reaction is run for about 1 minute to about 60 minutes. Insome embodiments, a sulfurization reaction is run for about 1 minute toabout 30 minutes. In some embodiments, a sulfurization reaction is runfor about 1 minute to about 25 minutes. In some embodiments, asulfurization reaction is run for about 1 minute to about 20 minutes. Insome embodiments, a sulfurization reaction is run for about 1 minute toabout 15 minutes. In some embodiments, a sulfurization reaction is runfor about 1 minute to about 10 minutes. In some embodiments, asulfurization reaction is run for about 5 minute to about 60 minutes.

In some embodiments, a sulfurization reaction is run for about 5minutes. In some embodiments, a sulfurization reaction is run for about10 minutes. In some embodiments, a sulfurization reaction is run forabout 15 minutes. In some embodiments, a sulfurization reaction is runfor about 20 minutes. In some embodiments, a sulfurization reaction isrun for about 25 minutes. In some embodiments, a sulfurization reactionis run for about 30 minutes. In some embodiments, a sulfurizationreaction is run for about 35 minutes. In some embodiments, asulfurization reaction is run for about 40 minutes. In some embodiments,a sulfurization reaction is run for about 45 minutes. In someembodiments, a sulfurization reaction is run for about 50 minutes. Insome embodiments, a sulfurization reaction is run for about 55 minutes.In some embodiments, a sulfurization reaction is run for about 60minutes.

It was unexpectedly found that certain of the sulfurization modificationproducts made in accordance with methods of the present invention areunexpectedly stable. In some embodiments, it the unexpectedly stableproducts are phosphorothioate triesters. In some embodiments, theunexpectedly stable products are chirally controlled oligonucleotidescomprising one or more internucleotidic linkages having the structure offormula I-c.

One of skill in the relevant arts will recognize that sulfurizationmethods described herein and sulfurization reagents described herein arealso useful in the context of modifying H-phosphonate oligonucleotidessuch as those described in Wada II (WO2010/064146).

In some embodiments, the sulfurization reaction has a stepwisesulfurization efficiency that is at least about 80%, 85%, 90%, 95%, 96%,97%, or 98%. In some embodiments, the sulfurization reaction provides acrude dinucleotide product composition that is at least 98% pure. Insome embodiments, the sulfurization reaction provides a crudetetranucleotide product composition that is at least 90% pure. In someembodiments, the sulfurization reaction provides a crudedodecanucleotide product composition that is at least 70% pure. In someembodiments, the sulfurization reaction provides a crude icosanucleotideproduct composition that is at least 50% pure.

Once the step of modifying the linkage phosphorus is complete, theoligonucleotide undergoes another deblock step in preparation forre-entering the cycle. In some embodiments, a chiral auxiliary remainsintact after sulfurization and is deblocked during the subsequentdeblock step, which necessarily occurs prior to re-entering the cycle.The process of deblocking, coupling, capping, and modifying, arerepeated until the growing oligonucleotide reaches a desired length, atwhich point the oligonucleotide can either be immediately cleaved fromthe solid support or left attached to the support for purificationpurposes and later cleaved. In some embodiments, one or more protectinggroups are present on one or more of the nucleotide bases, and cleavageof the oligonucleotide from the support and deprotection of the basesoccurs in a single step. In some embodiments, one or more protectinggroups are present on one or more of the nucleotide bases, and cleavageof the oligonucleotide from the support and deprotection of the basesoccurs in more than one steps. In some embodiments, deprotection andcleavage from the support occurs under basic conditions using, e.g., oneor more amine bases. In certain embodiments, the one or more amine basescomprise propyl amine. In certain embodiments, the one or more aminebases comprise pyridine.

In some embodiments, cleavage from the support and/or deprotectionoccurs at elevated temperatures of about 30° C. to about 90° C. In someembodiments, cleavage from the support and/or deprotection occurs atelevated temperatures of about 40° C. to about 80° C. In someembodiments, cleavage from the support and/or deprotection occurs atelevated temperatures of about 50° C. to about 70° C. In someembodiments, cleavage from the support and/or deprotection occurs atelevated temperatures of about 60° C. In some embodiments, cleavage fromthe support and/or deprotection occurs at ambient temperatures.

Exemplary purification procedures are described herein and/or are knowngenerally in the relevant arts.

Noteworthy is that the removal of the chiral auxiliary from the growingoligonucleotide during each cycle is beneficial for at least the reasonsthat (1) the auxiliary will not have to be removed in a separate step atthe end of the oligonucleotide synthesis when potentially sensitivefunctional groups are installed on phosphorus; and (2) unstablephosphorus-auxiliary intermediates prone to undergoing side reactionsand/or interfering with subsequent chemistry are avoided. Thus, removalof the chiral auxiliary during each cycle makes the overall synthesismore efficient.

While the step of deblocking in the context of the cycle is describedabove, additional general methods are included below.

Deblocking Step

In some embodiments, the step of coupling is preceded by a step ofdeblocking. For instance, in some embodiments, the 5′ hydroxyl group ofthe growing oligonucleotide is blocked (i.e., protected) and must bedeblocked in order to subsequently react with a nucleoside couplingpartner.

In some embodiments, acidification is used to remove a blocking group.In some embodiments, the acid is a Brønsted acid or Lewis acid. UsefulBrønsted acids are carboxylic acids, alkylsulfonic acids, arylsulfonicacids, phosphoric acid and its derivatives, phosphonic acid and itsderivatives, alkylphosphonic acids and their derivatives, arylphosphonicacids and their derivatives, phosphinic acid, dialkylphosphinic acids,and diarylphosphinic acids which have a pKa (25° C. in water) value of−0.6 (trifluoroacetic acid) to 4.76 (acetic acid) in an organic solventor water (in the case of 80% acetic acid). The concentration of the acid(1 to 80%) used in the acidification step depends on the acidity of theacid. Consideration to the acid strength must be taken into account asstrong acid conditions will result in depurination/depyrimidination,wherein purinyl or pyrimidinyl bases are cleaved from ribose ring and orother sugar ring. In some embodiments, an acid is selected fromR^(a1)COOH, R^(a1)SO₃H, R^(a3)SO₃H,

wherein each of R^(a1) and R^(a2) is independently hydrogen or anoptionally substituted alkyl or aryl, and R^(a3) is an optionallysubstituted alkyl or aryl.

In some embodiments, acidification is accomplished by a Lewis acid in anorganic solvent. Exemplary such useful Lewis acids are Zn(X^(a))₂wherein X^(a) is Cl, Br, I, or CF₃SO₃.

In some embodiments, the step of acidifying comprises adding an amountof a Brønsted or Lewis acid effective to remove a blocking group withoutremoving purine moieties from the condensed intermediate.

Acids that are useful in the acidifying step also include, but are notlimited to 10% phosphoric acid in an organic solvent, 10% hydrochloricacid in an organic solvent, 1% trifluoroacetic acid in an organicsolvent, 3% dichloroacetic acid or trichloroacetic acid in an organicsolvent or 80% acetic acid in water. The concentration of any Brønstedor Lewis acid used in this step is selected such that the concentrationof the acid does not exceed a concentration that causes cleavage of anucleobase from a sugar moiety.

In some embodiments, acidification comprises adding 1% trifluoroaceticacid in an organic solvent. In some embodiments, acidification comprisesadding about 0.1% to about 8% trifluoroacetic acid in an organicsolvent. In some embodiments, acidification comprises adding 3%dichloroacetic acid or trichloroacetic acid in an organic solvent. Insome embodiments, acidification comprises adding about 0.1% to about 10%dichloroacetic acid or trichloroacetic acid in an organic solvent. Insome embodiments, acidification comprises adding 3% trichloroacetic acidin an organic solvent. In some embodiments, acidification comprisesadding about 0.1% to about 10% trichloroacetic acid in an organicsolvent. In some embodiments, acidification comprises adding 80% aceticacid in water. In some embodiments, acidification comprises adding about50% to about 90%, or about 50% to about 80%, about 50% to about 70%,about 50% to about 60%, about 70% to about 90% acetic acid in water. Insome embodiments, the acidification comprises the further addition ofcation scavengers to an acidic solvent. In certain embodiments, thecation scavengers can be triethylsilane or triisopropylsilane. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 1% trifluoroacetic acid in an organic solvent. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 3% dichloroacetic acid in an organic solvent. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 3% trichloroacetic acid in an organic solvent. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 3% trichloroacetic acid in dichloromethane.

In certain embodiments, methods of the present invention are completedon a synthesizer and the step of deblocking the hydroxyl group of thegrowing oligonucleotide comprises delivering an amount solvent to thesynthesizer column, which column contains a solid support to which theoligonucleotide is attached. In some embodiments, the solvent is ahalogenated solvent (e.g., dichloromethane). In certain embodiments, thesolvent comprises an amount of an acid. In some embodiments, the solventcomprises an amount of an organic acid such as, for instance,trichloroacetic acid. In certain embodiments, the acid is present in anamount of about 1% to about 20% w/v. In certain embodiments, the acid ispresent in an amount of about 1% to about 10% w/v. In certainembodiments, the acid is present in an amount of about 1% to about 5%w/v. In certain embodiments, the acid is present in an amount of about 1to about 3% w/v. In certain embodiments, the acid is present in anamount of about 3% w/v. Methods for deblocking a hydroxyl group aredescribed further herein. In some embodiments, the acid is present in 3%w/v is dichloromethane.

In some embodiments, the chiral auxiliary is removed before thedeblocking step. In some embodiments, the chiral auxiliary is removedduring the deblocking step.

In some embodiments, cycle exit is performed before the deblocking step.In some embodiments, cycle exit is preformed after the deblocking step.

General Conditions for Blocking Group/Protecting Group Removal

Functional groups such as hydroxyl or amino moieties which are locatedon nucleobases or sugar moieties are routinely blocked with blocking(protecting) groups (moieties) during synthesis and subsequentlydeblocked. In general, a blocking group renders a chemical functionalityof a molecule inert to specific reaction conditions and can later beremoved from such functionality in a molecule without substantiallydamaging the remainder of the molecule (see e.g., Green and Wuts,Protective Groups in Organic Synthesis, 2nd Ed., John Wiley & Sons, NewYork, 1991). For example, amino groups can be blocked with nitrogenblocking groups such as phthalimido, 9-fludrenylmethoxycarbonyl (FMOC),triphenylmethylsulfenyl, t-BOC, 4,4′-dimethoxytrityl (DMTr),4-methoxytrityl (MMTr), 9-phenylxanthin-9-yl (Pixyl), trityl (Tr), or9-(p-methoxyphenyl)xanthin-9-yl (MOX). Carboxyl groups can be protectedas acetyl groups. Hydroxy groups can be protected such astetrahydropyranyl (THP), t-butyldimethylsilyl (TBDMS),1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (Ctmp),1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp),1-(2-chloroethoxy)ethyl, 3-methoxy-1,5-dicarbomethoxypentan-3-yl (MDP),bis(2-acetoxyethoxy)methyl (ACE), triisopropylsilyloxymethyl (TOM),1-(2-cyanoethoxy)ethyl (CEE), 2-cyanoethoxymethyl (CEM),[4-(N-dichloroacetyl-N-methylamino)benzyloxy]methyl, 2-cyanoethyl (CN),pivaloyloxymethyl (PivOM), levunyloxymethyl (ALE). Other representativehydroxyl blocking groups have been described (see e.g., Beaucage et al.,Tetrahedron, 1992, 46, 2223). In some embodiments, hydroxyl blockinggroups are acid-labile groups, such as the trityl, monomethoxytrityl,dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and9-(p-methoxyphenyl)xanthin-9-yl (MOX). Chemical functional groups canalso be blocked by including them in a precursor form. Thus an azidogroup can be considered a blocked form of an amine as the azido group iseasily converted to the amine. Further representative protecting groupsutilized in nucleic acid synthesis are known (see e.g. Agrawal et al.,Protocols for Oligonucleotide Conjugates, Eds., Humana Press, NewJersey, 1994, Vol. 26, pp. 1-72).

Various methods are known and used for removal of blocking groups fromnucleic acids. In some embodiments, all blocking groups are removed. Insome embodiments, a portion of blocking groups are removed. In someembodiments, reaction conditions can be adjusted to selectively removecertain blocking groups.

In some embodiments, nucleobase blocking groups, if present, arecleavable with an acidic reagent after the assembly of a providedoligonucleotide. In some embodiment, nucleobase blocking groups, ifpresent, are cleavable under neither acidic nor basic conditions, e.g.cleavable with fluoride salts or hydrofluoric acid complexes. In someembodiments, nucleobase blocking groups, if present, are cleavable inthe presence of base or a basic solvent after the assembly of a providedoligonucleotide. In certain embodiments, one or more of the nucleobaseblocking groups are characterized in that they are cleavable in thepresence of base or a basic solvent after the assembly of a providedoligonucleotide but are stable to the particular conditions of one ormore earlier deprotection steps occurring during the assembly of theprovided oligonucleotide.

In some embodiments, blocking groups for nucleobases are not required.In some embodiments, blocking groups for nucleobases are required. Insome embodiments, certain nucleobases require one or more blockinggroups while other nucleobases do not require one or more blockinggroups.

In some embodiments, the oligonucleotide is cleaved from the solidsupport after synthesis. In some embodiments, cleavage from the solidsupport comprises the use of propylamine. In some embodiments, cleavagefrom the solid support comprises the use of propylamine in pyridine. Insome embodiments, cleavage from the solid support comprises the use of20% propylamine in pyridine. In some embodiments, cleavage from thesolid support comprises the use of propylamine in anhydrous pyridine. Insome embodiments, cleavage from the solid support comprises the use of20% propylamine in anhydrous pyridine. In some embodiments, cleavagefrom the solid support comprises use of a polar aprotic solvent such asacetonitrile, NMP, DMSO, sulfone, and/or lutidine. In some embodiments,cleavage from the solid support comprises use of solvent, e.g., a polaraprotic solvent, and one or more primary amines (e.g., a C₁₋₁₀ amine),and/or one or more of methoxylamine, hydrazine, and pure anhydrousammonia.

In some embodiments, deprotection of oligonucleotide comprises the useof propylamine. In some embodiments, deprotection of oligonucleotidecomprises the use of propylamine in pyridine. In some embodiments,deprotection of oligonucleotide comprises the use of 20% propylamine inpyridine. In some embodiments deprotection of oligonucleotide comprisesthe use of propylamine in anhydrous pyridine. In some embodiments,deprotection of oligonucleotide comprises the use of 20% propylamine inanhydrous pyridine.

In some embodiments, the oligonucleotide is deprotected during cleavage.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at about room temperature.In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at elevated temperature.In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at above about 30° C., 40°C., 50° C., 60° C., 70° C., 80° C. 90° C. or 100° C. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at about 30° C., 40° C.,50° C., 60° C., 70° C., 80° C. 90° C. or 100° C. In some embodiments,cleavage of oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at about 40-80° C. In some embodiments,cleavage of oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at about 50-70° C. In some embodiments,cleavage of oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at about 60° C.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for more than 0.1 hr, 1hr, 2 hrs, 5 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, 30 hrs, or 40 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 0.1-5 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 3-10 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 5-15 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 10-20 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 15-25 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 20-40 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 2 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 5 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 10 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 15 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 18 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 24 hrs.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at room temperature formore than 0.1 hr, 1 hr, 2 hrs, 5 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, 30hrs, or 40 hrs. In some embodiments, cleavage of oligonucleotide fromsolid support, or deprotection of oligonucleotide, is performed at roomtemperature for about 5-48 hrs. In some embodiments, cleavage ofoligonucleotide from solid support, or deprotection of oligonucleotide,is performed at room temperature for about 10-24 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at room temperature forabout 18 hrs. In some embodiments, cleavage of oligonucleotide fromsolid support, or deprotection of oligonucleotide, is performed atelevated temperature for more than 0.1 hr, 1 hr, 2 hrs, 5 hrs, 10 hrs,15 hrs, 20 hrs, 24 hrs, 30 hrs, or 40 hrs. In some embodiments, cleavageof oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at elevated temperature for about 0.5-5hrs. In some embodiments, cleavage of oligonucleotide from solidsupport, or deprotection of oligonucleotide, is performed at about 60°C. for about 0.5-5 hrs. In some embodiments, cleavage of oligonucleotidefrom solid support, or deprotection of oligonucleotide, is performed atabout 60° C. for about 2 hrs.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide comprises the use of propylamine and isperformed at room temperature or elevated temperature for more than 0.1hr, 1 hr, 2 hrs, 5 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, 30 hrs, or 40hrs. Exemplary conditions are 20% propylamine in pyridine at roomtemperature for about 18 hrs, and 20% propylamine in pyridine at 60° C.for about 18 hrs,

In some embodiments, the present invention provides methods for making achirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved;

wherein the chirally controlled oligonucleotide comprises at least onephosphorothioate diester linkage or at least one internucleotidiclinkage having the structure of formula I-c.

In some embodiments, the present invention provides methods for making achirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved;

wherein:at least one cycle of (1) to (4) forms an phosphorothioate diesterlinkage.

In some embodiments, the present invention provides methods for making achirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved;

wherein:at least one cycle of (1) to (4) forms an internucleotidic linkagehaving the structure of formula I-c.

In some embodiments, the present invention provides methods for making achirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved;

wherein:

-   the coupling step comprises the use of an activating group and

wherein B^(PRO) is a protected nucleobase;

-   the capping step comprising capping of the amino group in the chiral    auxiliary and the capping of unreacted 5′-OH;-   the modifying step comprising installation of —S-L-R¹ group to the    linkage phosphorus, wherein each of L and R¹ is independently as    defined above and described herein;-   the delocking step comprising the use of an acid.

In some embodiments, the present invention provides methods for making achirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved;

wherein:

-   the coupling step comprises the use of CMPT and

wherein B^(PRO) is a protected nucleobase;

-   the capping step comprising capping of the amino group in the chiral    auxiliary and the capping of unreacted 5′-OH;-   the modifying step comprising installation of —S-L-R¹ group to the    linkage phosphorus, wherein each of L and R¹ is independently as    defined above and described herein;-   the deblocking step comprising the use of an acid.

In some embodiments, an activator is a “Wada” activator, i.e., theactivator is from any one of Wada I, II, or III documents cited above.

Exemplary activating groups are depicted below:

An exemplary cycle is depicted in Scheme I-b.

In some embodiments, the present invention provides methods for making achirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved;

wherein:the chirally controlled oligonucleotide comprises at least onephosphorothioate diester linkage or at least one internucleotidiclinkage of formula I-c, and at least one phosphate diesterinternucleotidic linkage; andat least one modifying step is replaced by an oxidization step.

In some embodiments, the present invention provides methods for making achirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved;

wherein:the chirally controlled oligonucleotide comprises at least onephosphorothioate diester linkage or at least one internucleotidiclinkage of formula I-c, and at least one phosphate diesterinternucleotidic linkage; andat least one modifying step is replaced by an oxidization stepcomprising the use of I₂.

An exemplary cycle is illustrated in Scheme I-c.

In Scheme I-c, oligonucleotide (or nucleotide, or oligonucleotide withmodified internucleotidic linkage) on solid support (C-1) is coupledwith phosphoramidite C-2. After coupling and capping, an oxidation stepis performed. After deblocking, a phosphate diester linkage is formed.The cycle product C-3 can either re-enter cycle C to install morephosphate diester linkage, or enter other cycles to install other typesof internucleotidic linkages, or go to cycle exit.

In some embodiments, non-chirally pure phosphoramidite can be usedinstead of C-2 in Scheme I-c. In some embodiments,β-cyanoethylphosphoramidites protected with DMTr is used. In someembodiments, the phosphoramidite being used has the structure of

In some embodiments, the present invention provides methods for making achirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved;

wherein:the chirally controlled oligonucleotide comprises one or morephosphorothioate diester linkages; andone or more phosphorothioate diester precursors are formed for each ofthe corresponding phosphorothioate diester linkage.

In some embodiments, the present invention provides methods for making achirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved;

wherein:the chirally controlled oligonucleotide comprises at least onephosphorothioate diester linkages; one or more phosphorothioate diesterprecursors are formed for each of the corresponding phosphorothioatediester linkage; andeach phosphorothioate diester precursor is converted to aphosphorothioate diester linkage after the desired length is achieved.

In some embodiments, the present invention provides methods for making achirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved;

wherein:the chirally controlled oligonucleotide comprises at least onephosphorothioate diester linkages and at least one phosphate diesterinternucleotidic linkage;at least one modifying step is replaced by an oxidization step; andat least one modifying step is performed to install a phosphorothioatediester precursor for each of the phosphorothioate diester linkages; andeach phosphorothioate diester precursor is converted to aphosphorothioate diester linkage after the desired length is achieved.

In some embodiments, the use of a phosphorothioate diester precursorincreases the stability of oligonucleotide during synthesis. In someembodiments, the use of a phosphorothioate diester precursor improvesthe efficiency of chirally controlled oligonucleotide synthesis. In someembodiments, the use of a phosphorothioate diester precursor improvesthe yield of chirally controlled oligonucleotide synthesis. In someembodiments, the use of a phosphorothioate diester precursor improvesthe product purity of chirally controlled oligonucleotide synthesis.

In some embodiments, the phosphorothioate diester precursor in theabove-mentioned methods is

In some embodiments,

is converted to a phosphorothioate diester linkage duringdeprotection/release. An exemplary cycle is depicted in Scheme I-d. Moreexamples are depicted below.

As illustrated in Scheme I-d, both phosphorothioate and phosphatediester linkages can be incorporated into the same chirally controlledoligonucleotide. As understood by a person of ordinary skill in the art,the provided methods do not require that the phosphorothioate diesterand the phosphate diester to be consecutive—other internucleotidiclinkages can form between them using a cycle as described above. InScheme I-d, phosphorothioate diester precursors,

are installed in place of the phosphorothioate diester linkages. In someembodiments, such replacement provided increased synthesis efficiencyduring certain steps, for instance, the oxidation step. In someembodiments, the use of phosphorothioate diester precursors generallyimprove the stability of chirally controlled oligonucleotides duringsynthesis and/or storage. After cycle exit, during deprotection/release,the phosphorothioate diester precursor is converted to phosphorothioatediester linkage. In some embodiments, it is beneficial to usephosphorothioate diester precursor even when no phosphate diesterlinkage is present in the chirally controlled oligonucleotide, or nooxidation step is required during synthesis.

As in Scheme I-c, in some embodiments, non-chirally pure phosphoramiditecan be used for cycles comprising oxidation steps. In some embodiments,β-cyanoethylphosphoramidites protected with DMTr is used. In someembodiments, the phosphoramidite being used has the structure of

In some embodiments, methods of the present invention provide chirallycontrolled oligonucleotide compositions that are enriched in aparticular oligonucleotide type.

In some embodiments, at least about 10% of a provided crude compositionis of a particular oligonucleotide type. In some embodiments, at leastabout 20% of a provided crude composition is of a particularoligonucleotide type. In some embodiments, at least about 30% of aprovided crude composition is of a particular oligonucleotide type. Insome embodiments, at least about 40% of a provided crude composition isof a particular oligonucleotide type. In some embodiments, at leastabout 50% of a provided crude composition is of a particularoligonucleotide type. In some embodiments, at least about 60% of aprovided crude composition is of a particular oligonucleotide type. Insome embodiments, at least about 70% of a provided crude composition isof a particular oligonucleotide type. In some embodiments, at leastabout 80% of a provided crude composition is of a particularoligonucleotide type. In some embodiments, at least about 90% of aprovided crude composition is of a particular oligonucleotide type. Insome embodiments, at least about 95% of a provided crude composition isof a particular oligonucleotide type.

In some embodiments, at least about 1% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 2%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 3% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 4%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 5% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 10%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 20% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 30%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 40% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 50%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 60% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 70%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 80% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 90%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 95% of a provided composition is of aparticular oligonucleotide type.

Biological Applications

As discussed in detail herein, the present invention provides, amongother things, a chirally controlled oligonucleotide composition, meaningthat the composition contains a plurality of oligonucleotides of atleast one type. Each oligonucleotide molecule of a particular “type” iscomprised of preselected (e.g., predetermined) structural elements withrespect to: (1) base sequence; (2) pattern of backbone linkages; (3)pattern of backbone chiral centers; and (4) pattern of backboneP-modification moieties. In some embodiments, provided oligonucleotidecompositions contain oligonucleotides that are prepared in a singlesynthesis process. In some embodiments, provided compositions containoligonucleotides having more than one chiral configuration within asingle oligonucleotide molecule (e.g., where different residues alongthe oligonucleotide have different stereochemistry); in some suchembodiments, such oligonucleotides may be obtained in a single synthesisprocess, without the need for secondary conjugation steps to generateindividual oligonucleotide molecules with more than one chiralconfiguration.

Oligonucleotide compositions as provided herein can be used as agentsfor modulating a number of cellular processes and machineries, includingbut not limited to, transcription, translation, immune responses,epigenetics, etc. In addition, oligonucleotide compositions as providedherein can be used as reagents for research and/or diagnostic purposes.One of ordinary skill in the art will readily recognize that the presentinvention disclosure herein is not limited to particular use but isapplicable to any situations where the use of synthetic oligonucleitidesis desirable. Among other things, provided compositions are useful in avariety of therapeutic, diagnostic, agricultural, and/or researchapplications.

In some embodiments, provided oligonucleotide compositions compriseoligonucleotides and/or residues thereof that include one or morestructural modifications as described in detail herein. In someembodiments, provided oligonucleotide compositions compriseoligonucleoties that contain one or more nucleic acid analogs. In someembodiments, provided oligonucleotide compositions compriseoligonucleotides that contain one or more artificial nucleic acids orresidues, including but not limited to: peptide nucleic acids (PNA),Morpholino and locked nucleic acids (LNA), glycon nucleic acids (GNA),threose nucleic acids (TNA), Xeno nucleic acids (ZNA), and anycombination thereof.

In any of the embodiments, the invention is useful foroligonucleotide-based modulation of gene expression, immune response,etc. Accordingly, stereo-defined, oligonucleotide compositions of theinvention, which contain oligonucleotides of predetermined type (i.e.,which are chirally controlled, and optionally chirally pure), can beused in lieu of conventional stereo-random or chirally impurecounterparts. In some embodiments, provided compositions show enhancedintended effects and/or reduced unwanted side effects. Certainembodiments of biological and clinical/therapeutic applications of theinvention are discussed explicitly below.

Various dosing regimens can be utilized to administer provided chirallycontrolled oligonucleotide compositions. In some embodiments, multipleunit doses are administered, separated by periods of time. In someembodiments, a given composition has a recommended dosing regimen, whichmay involve one or more doses. In some embodiments, a dosing regimencomprises a plurality of doses each of which are separated from oneanother by a time period of the same length; in some embodiments, adosing regimen comprises a plurality of doses and at least two differenttime periods separating individual doses. In some embodiments, all doseswithin a dosing regimen are of the same unit dose amount. In someembodiments, different doses within a dosing regimen are of differentamounts. In some embodiments, a dosing regimen comprises a first dose ina first dose amount, followed by one or more additional doses in asecond dose amount different from the first dose amount. In someembodiments, a dosing regimen comprises a first dose in a first doseamount, followed by one or more additional doses in a second (orsubsequent) dose amount that is same as or different from the first dose(or another prior dose) amount. In some embodiments, a dosing regimencomprises administering at least one unit dose for at least one day. Insome embodiments, a dosing regimen comprises administering more than onedose over a time period of at least one day, and sometimes more than oneday. In some embodiments, a dosing regimen comprises administeringmultiple doses over a time period of at least week. In some embodiments,the time period is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100 or more) weeks. In some embodiments, adosing regimen comprises administering one dose per week for more thanone week. In some embodiments, a dosing regimen comprises administeringone dose per week for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100 or more) weeks. In some embodiments, a dosingregimen comprises administering one dose every two weeks for more thantwo week period. In some embodiments, a dosing regimen comprisesadministering one dose every two weeks over a time period of 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more(e.g., about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more)weeks. In some embodiments, a dosing regimen comprises administering onedose per month for one month. In some embodiments, a dosing regimencomprises administering one dose per month for more than one month. Insome embodiments, a dosing regimen comprises administering one dose permonth for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In someembodiments, a dosing regimen comprises administering one dose per weekfor about 10 weeks. In some embodiments, a dosing regimen comprisesadministering one dose per week for about 20 weeks. In some embodiments,a dosing regimen comprises administering one dose per week for about 30weeks. In some embodiments, a dosing regimen comprises administering onedose per week for 26 weeks. In some embodiments, a chirally controlledoligonucleotide composition is administered according to a dosingregimen that differs from that utilized for a chirally uncontrolled(e.g., stereorandom) oligonucleotide composition of the same sequence,and/or of a different chirally controlled oligonucleotide composition ofthe same sequence. In some embodiments, a chirally controlledoligonucleotide composition is administered according to a dosingregimen that is reduced as compared with that of a chirally uncontrolled(e.g., stereorandom) oligonucleotide composition of the same sequence inthat it achieves a lower level of total exposure over a given unit oftime, involves one or more lower unit doses, and/or includes a smallernumber of doses over a given unit of time. In some embodiments, achirally controlled oligonucleotide composition is administeredaccording to a dosing regimen that extends for a longer period of timethan does that of a chirally uncontrolled (e.g., stereorandom)oligonucleotide composition of the same sequence Without wishing to belimited by theory, Applicant notes that in some embodiments, the shorterdosing regimen, and/or longer time periods between doses, may be due tothe improved stability, bioavailability, and/or efficacy of a chirallycontrolled oligonucleotide composition. In some embodiments, a chirallycontrolled oligonucleotide composition has a longer dosing regimencompared to the corresponding chirally uncontrolled oligonucleotidecomposition. In some embodiments, a chirally controlled oligonucleotidecomposition has a shorter time period between at least two dosescompared to the corresponding chirally uncontrolled oligonucleotidecomposition. Without wishing to be limited by theory, Applicant notesthat in some embodiments longer dosing regimen, and/or shorter timeperiods between doses, may be due to the improved safety of a chirallycontrolled oligonucleotide composition.

A single dose can contain various amounts of a type of chirallycontrolled oligonucleotide, as desired suitable by the application. Insome embodiments, a single dose contains about 1, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more (e.g., about350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 ormore) mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 1 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 5 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 10 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 15 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 20 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 50 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 100 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 150 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 200 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 250 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 300 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a chirally controlledoligonucleotide is administered at a lower amount in a single dose,and/or in total dose, than a chirally uncontrolled oligonucleotide. Insome embodiments, a chirally controlled oligonucleotide is administeredat a lower amount in a single dose, and/or in total dose, than achirally uncontrolled oligonucleotide due to improved efficacy. In someembodiments, a chirally controlled oligonucleotide is administered at ahigher amount in a single dose, and/or in total dose, than a chirallyuncontrolled oligonucleotide. In some embodiments, a chirally controlledoligonucleotide is administered at a higher amount in a single dose,and/or in total dose, than a chirally uncontrolled oligonucleotide dueto improved safety.

Biologically Active Oligonucleotides

A provided oligonucleotide composition as used herein may comprisesingle stranded and/or multiply stranded oligonucleotides. In someembodiments, single-stranded oligonucleotides contain self-complementaryportions that may hybridize under relevant conditions so that, as used,even single-stranded oligonucleotides may have at least partiallydouble-stranded character. In some embodiments, an oligonucleotideincluded in a provided composition is single-stranded, double-stranded,or triple-stranded. In some embodiments, an oligonucleotide included ina provided composition comprises a single-stranded portion and amultiple-stranded portion within the oligonucleotide. In someembodiments, as noted above, individual single-stranded oligonucleotidescan have double-stranded regions and single-stranded regions.

In some embodiments, provided compositions include one or moreoligonucleotides fully or partially complementary to strand of:structural genes, genes control and/or termination regions, and/orself-replicating systems such as viral or plasmid DNA. In someembodiments, provided compositions include one or more oligonucleotidesthat are or act as siRNAs or other RNA interference reagents (RNAiagents or iRNA agents), shRNA, antisense oligonucleotides, self-cleavingRNAs, ribozymes, fragment thereof and/or variants thereof (such asPeptidyl transferase 23S rRNA, RNase P, Group I and Group II introns,GIR1 branching ribozymes, Leadzyme, Hairpin ribozymes, Hammerheadribozymes, HDV ribozymes, Mammalian CPEB3 ribozyme, VS ribozymes, glmSribozymes, CoTC ribozyme, etc.), microRNAs, microRNA mimics, supermirs,aptamers, antimirs, antagomirs, Ul adaptors, triplex-formingoligonucleotides, RNA activators, long non-coding RNAs, short non-codingRNAs (e.g., piRNAs), immunomodulatory oligonucleotides (such asimmunostimulatory oligonucleotides, immunoinhibitory oligonucleotides),GNA, LNA, ENA, PNA, TNA, morpholinos, G-quadruplex (RNA and DNA),antiviral oligonucleotides, and decoy oligonucleotides.

In some embodiments, provided compositions include one or more hybrid(e.g., chimeric) oligonucleotides. In the context of the presentdisclosure, the term “hybrid” broadly refers to mixed structuralcomponents of oligonucleotides. Hybrid oligonucleotides may refer to,for example, (1) an oligonucleotide molecule having mixed classes ofnucleotides, e.g., part DNA and part RNA within the single molecule(e.g., DNA-RNA); (2) complementary pairs of nucleic acids of differentclasses, such that DNA:RNA base pairing occurs either intramolecularlyor intermolecularly; or both; (3) an oligonucleotide with two or morekinds of the backbone or internucleotide linkages.

In some embodiments, provided compositions include one or moreoligonucleotide that comprises more than one classes of nucleic acidresidues within a single molecule. For example, in any of theembodiments described herein, an oligonucleotide may comprise a DNAportion and an RNA portion. In some embodiments, an oligonucleotide maycomprise a unmodified portion and modified portion.

Provided oligonucleotide compositions can include oligonucleotidescontaining any of a variety of modifications, for example as describedherein. In some embodiments, particular modifications are selected, forexample, in light of intended use. In some embodiments, it is desirableto modify one or both strands of a double-stranded oligonucleotide (or adouble-stranded portion of a single-stranded oligonucleotide). In someembodiments, the two strands (or portions) include differentmodifications. In some embodiments, the two strands include the samemodifications. One of skill in the art will appreciate that the degreeand type of modifications enabled by methods of the present inventionallow for numerous permutations of modifications to be made. Exemplarysuch modifications are described herein and are not meant to belimiting.

RNA Interference

Provided oligonucleotide compositions are useful, among other things,for applications in RNA interference.

RNA interference (RNAi) refers to the inhibition of gene expression byRNA molecules. Typically, these are small, double-stranded RNAmolecules. Since gene expression controls most cellular processes, theability to inhibit gene expression provides a potentially powerful toolfor modulating biological conditions, including treating human and/oranimal (e.g., livestock or pet) diseases. A number of studies have beenconducted to demonstrate the use of RNAi in regulating or controllingdisease-associated gene expression. See, for example: Cullen, K. A.,Hall, M. J. & Golosinskiy, A. Ambulatory surgery in the United States,2006. Natl Health Stat Report 2009; 1-25; Elbashir S, Harborth J,Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells. Nature 2001; 411:494-498; Fire A, Xu S, Montgomery M K, Kostas S A, Driveri S E & MelloC. Potent and specific RNA interference by double-stranded RNA inCaenorhadbditis elegans. Nature 1998; 391 (6669):806-811; Gauglitz, G.G., Kortin, H. C., Pavicic, T., Ruzicka, T., & Jeschke, M. G.Hypertrophic scarring and keloids: pathomechanisms and current emergingtreatment strategies. Mol Med 2011; 17(1-2): 113-125; Li-Tsang, C. W.,Lau, J. C. & Chan, C. C. Prevalence of hypertrophic scar formation andits characteristics among the Chinese population. Burns 2005; 31,610-616; Wang H, Ghosh A, Baigude H, Yang C, Qui L, Xia L, et al.Therapeutic gene silencing delivered by a chemically modified siRNAagainst mutant SOD1 slows ALS progression. JBC 2008; 283(23):15845-15852; Weiser, T. G., Regenbogen, S. E., Thompson, K. D.,Haynes, A. B., Lipsitz, S. R., Berry, W. R. & Gawande, A. A. Anestimation of the global volume of surgery: a modeling strategy based onavailable data. Lancet 2008; 372(9633): 139-44.

The phenomenon of RNA interference was initially demonstrated in C.elegans, in which the injection of dsRNA molecules inhibitedcomplementary gene expression. Though the use of siRNA has become awidely used tool for down-regulating gene expression, the existence of anaturally occurring pathway in eukaryotes has been well described. Theorigin of endogenous siRNA (or miRNA) may be transposons, viruses,repetitive sequences and genes. The process of producing effectiveendogenous siRNA is regulated by three enzymes. RNA-dependent RNApolymerases convert single-stranded RNA into double-stranded RNA.Alternatively, DNA-dependent RNA polymerases produce dsRNA bytranscribing inverted DNA repeats. The resulting large RNA molecules aresubject to digestion by ribonuclease III (Dicer) to produce shortdouble-stranded siRNA molecules. Argonaute proteins are then required tobind siRNA molecules to form a complex known as RISC (RNA-inducedsilencing complex). RISC recognizes double-stranded RNA fragment andsplits the double-strands apart, retaining one strand in the RISCcomplex. RISCs may then promote epigentic silencing through RNA-directedDNA methylation or by target RNA cleavage. Though protein translationmay be knocked down considerably, siRNA does not normally eliminate theexpression of a gene target completely. RISC can therefore help theguide strand of RNA bind to and destroy its corresponding cellularmessenger RNA target. Thus, RNAi provides a method to potentially blockthe creation of the proteins that cause disease.

siRNA technology represents a useful molecular tool. The use of RNAinterference for artificially manipulating gene expression was initiallylimited by the activation of cellular antiviral mechanisms. Exposure ofcells to sequences longer than 30 nucleotides has been shown to induceinterferon gene expression resulting in non-specific RNA degradation andreduced protein synthesis. However, this problem can be circumvented bydesigning short (e.g., 19 to 22 nucleotide) siRNA sequences. Methods forsiRNA delivery into cells include, without limitation, liposome-basedaddition of purified ribonucleotides to the media or transfection ofplasmid vectors designed to express siRNA molecules. Plasmid vectorsrely on the use of two RNA Polymerase III promoters (U6 and H1) to drivetranscription of the siRNA molecule. The target sequence (19 to 29nucleotides) is placed in a sense and antisense orientation with a smallspacer group in between (short hairpin RNA or shRNA). Once transcribed,a hairpin structure is formed that can be recognized and cleaved byDicer. Alternatively, RNA duplexes may be transcribed without hairpinstructures and directly process by the RISC. Currently, there are avariety of plasmid and viral vectors that utilize similar concepts toproduce siRNA, shRNA, or single stranded siRNA (ss-siRNA) molecules(See, e.g., 2012 Cell-150-883 Walt Lima et al. ssRNAi activate RNAi inanimals).

In some embodiments, a provided oligonucleotide or oligonucleotidecomposition is useful as ss-siRNA or GalNAc conjugated siRNA.

The art is familiar with certain structural features that affect siRNAas a tool. Following the discovery of siRNA, several studies attemptedto identify the optimal characteristics required for siRNA design. Someof the requirements include using sequences shorter than 30 nucleotidesto avoid PKR activation, sequence stability at the 5′ end of theantisense strand relative to the 3′ terminus and inserting a TToverhang. Based on studies like these, a number of algorithms have beendeveloped by academic and industrial labs to predict the most effectivetarget sequences for a given gene. Though most of these programs are notperfect, the likelihood of obtaining a predicted sequence is superior todesigning sequences without consideration of the recommended features.Synthesis and testing of multiple sequences may be required. The designof siRNA experiments may contain some potential pitfalls, thus thedesign should be done to include appropriate controls and measurableendpoints. A negative control may include a non-complementary sequencewith thermodynamically similar properties as the effective siRNAsequence. When transfecting a plasmid vector to introduce siRNA orshRNA, the ratio of lipid to nucleic acid may be equal and the controlvector may contain a sequence that is transcribed and processedintracellularly. Validation of the siRNA effect may also be carried outby measuring both RNA and protein expression.

In some embodiments, a provided oligonucleotide as used herein isdouble-stranded. Typically, double-stranded oligonucleotides comprisinga duplex structure of between 20 and 23, but specifically 21, base pairshave been hailed as particularly effective in inducing RNA interference(Elbashir ct al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer double-stranded oligonucleotides can be effectiveas well.

In some embodiments, a double-stranded oligonucleotide utilized inaccordance with the present invention comprises two oligonucleotidestrands that are sufficiently complementary to hybridize to form aduplex structure. In some embodiments, a duplex structure is betweenabout 12 to about 45 base pairs in length. In some embodiments, a duplexstructure is between about 18 to about 25 base pairs in length. In someembodiments, a duplex structure is between about 19 to about 24 basepairs in length. In some embodiments, a duplex structure is betweenabout 19 to about 21 base pairs in length. In some embodiments, a duplexstructure is a double-stranded oligonucleotides of between about 25 toabout 30 base pairs in length. In some embodiments, a duplex structureis a double-stranded oligonucleotide of between about 10 to about 15base pairs in length. In some embodiments, a double-strandedoligonucleotide is at least about 21 nucleotides long.

In some embodiments, a double-stranded oligonucleotide utilized inaccordance with the present invention comprises a sense strand and anantisense strand, wherein the antisense RNA strand has a region ofcomplementarity which is complementary to at least a part of a targetsequence, and the duplex region is about 14 to about 30 nucleotides inlength. In some embodiments, a region of complementarity to the targetsequence is between about 14 to about 30 nucleotides in length. In someembodiments, a region of complementarity to the target sequence isbetween about 18 to about 25 nucleotides in length. In some embodiments,a region of complementarity to the target sequence is between about 19to about 24 nucleotides in length. In some embodiments, a region ofcomplementarity to the target sequence is about 19 to about 21nucleotides in length.

The phrase “antisense strand” as used herein, refers to anoligonucleotide that is substantially or 100% complementary to a targetsequence of interest. The phrase “antisense strand” includes theantisense region of both oligonucleotides that are formed from twoseparate strands, as well as unimolecular oligonucleotides that arecapable of forming hairpin or dumbbell type structures. The terms“antisense strand” and “guide strand” are used interchangeably herein.

The phrase “sense strand” refers to an oligonucleotide that has the samenucleoside sequence, in whole or in part, as a target sequence such as amessenger RNA or a sequence of DNA. The terms “sense strand” and“passenger strand” are used interchangeably herein.

By “target sequence” is meant any nucleic acid sequence whose expressionor activity is to be modulated. The target nucleic acid can be DNA orRNA, such as endogenous DNA or RNA, viral DNA or viral RNA, or other RNAencoded by a gene, virus, bacteria, fungus, mammal, or plant. In someembodiments, a target sequence is associated with a disease or disorder.

By “specifically hybridizable” and “complementary” is meant that anucleic acid can form hydrogen bond(s) with another nucleic acidsequence by either traditional Watson-Crick or other non-traditionaltypes. In reference to the nucleic molecules of the present invention,the binding free energy for a nucleic acid molecule with itscomplementary sequence is sufficient to allow the relevant function ofthe nucleic acid to proceed, e.g., RNAi activity. Determination ofbinding free energies for nucleic acid molecules is well known in theart (see, e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LIT pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377;Turner et al., 1987, /. Ain. Chem. Soc. 109:3783-3785)

A percent complementarity indicates the percentage of contiguousresidues in a nucleic acid molecule that can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” or 100% complementarity meansthat all the contiguous residues of a nucleic acid sequence willhydrogen bond with the same number of contiguous residues in a secondnucleic acid sequence. Less than perfect complementarity refers to thesituation in which some, but not all, nucleoside units of two strandscan hydrogen bond with each other. “Substantial complementarity” refersto polynucleotide strands exhibiting 90% or greater complementarity,excluding regions of the polynucleotide strands, such as overhangs, thatare selected so as to be noncomplementary. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, e.g., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.In some embodiments, non-target sequences differ from correspondingtarget sequences by at least 5 nucleotides.

Double Stranded Oligonucleotides

In some embodiments, a double-stranded oligonucleotide utilized inaccordance with the present invention is sufficiently large that it canbe cleaved by an endogenous molecule, e.g., by Dicer, to produce smallerdouble-stranded oligonucleotides, e.g., RNAi agents. In someembodiments, a provided double-stranded oligonucleotide modulates theexpression of a target gene via RISC mediated cleavage of the targetsequence.

In some embodiments, a double-stranded region of a double-strandedoligonucleotide is equal to or at least, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotidepairs in length.

In some embodiments, an antisense strand of a double-strandedoligonucleotide is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, a sense strand of a double-stranded oligonucleotideis equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, one strand has at least one stretch of 1-5single-stranded nucleotides in the double-stranded region. By “stretchof single-stranded nucleotides in the double-stranded region” is meantthat there is present at least one nucleotide base pair at both ends ofthe single-stranded stretch. In some embodiments, both strands have atleast one stretch of 1-5 (e.g., 1, 2, 3, 4, or 5) single-strandednucleotides in the double stranded region. When both strands have astretch of 1-5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides inthe double stranded region, such single-stranded nucleotides can beopposite to each other (e.g., a stretch of mismatches) or they can belocated such that the second strand has no single-stranded nucleotidesopposite to the single-stranded oligonucleotides of the first strand andvice versa (e.g., a single-stranded loop). In some embodiments, thesingle-stranded nucleotides are present within 8 nucleotides from eitherend, for example 8, 7, 6, 5, 4, 3, or 2 nucleotide from either the 5′ or3′ end of the region of complementarity between the two strands.

In some embodiments, each strand of a double-stranded oligonucleotideutilized in accordance with the present invention has a ZXY structure,such as is described in International Application No. PCT/US2004/07070filed on Mar. 8, 2004, contents of which are hereby incorporated intheir entireties.

Hairpins and Dumbbells

In some embodiments, a double-stranded oligonucleotide utilized inaccordance with the present invention is a single molecule thatcomprises self-complementary regions; thus the two “strands” of adouble-stranded regions are in fact covalently linked to one another.Such two strands can be linked to each other at both ends, or at one endonly. By linking at one end is meant that 5′-end of first strand islinked to the 3′-end of the second strand or 3′-end of first strand islinked to 5′-end of the second strand. When the two strands are linkedto each other at both ends, 5′-end of first strand is linked to 3′-endof second strand and 3′-end of first strand is linked to 5′-end ofsecond strand. In some embodiments, two strands are linked together byan oligonucleotide linker including, but not limited to, (N)_(n);wherein N is independently a modified or unmodified nucleotide and n is3-23. In some embodiments, n is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10.In some embodiments, an oligonucleotide linker is selected from thegroup consisting of GNRA, (G)₄, (U)₄, and (dT)₄, wherein N is a modifiedor unmodified nucleotide and R is a modified or unmodified purinenucleotide. In some embodiments, some of the nucleotides in the linkerare involved in base-pair interactions with other nucleotides in thelinker. In some embodiments, the two strands are linked together by anon-nucleosidic linker, e.g.

In some embodiments, hairpin and dumbbell type RNAi agents have a duplexregion equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23,24, or 25 nucleotide pairs. In some embodiments, the duplex region isequal to or fewer than 200, 100, or 50, nucleotide pairs in length. Insome embodiments, ranges for the duplex region are about 15 to about 30,about 17 to about 23, about 19 to about 23, and about 19 to about 21nucleotides pairs in length. In some embodiments, hairpinoligonucleotides mimic the natural precursors of microRNAs.

In some embodiments, hairpin RNAi agents can have a single strandoverhang or terminal unpaired region, e.g., at the 3′ end on theantisense side of the hairpin, etc. In some embodiments, the overhangsare about 1 to about 4 nucleotides in length. In some embodiments, theoverhangs are about 2 to about 3 nucleotides in length.

In some embodiments, a hairpin RNAi agent is characterized in that the3′-end of an antisense strand is linked to 5′-end of a sense strand. Insome embodiments, a hairpin RNAi agent is characterized in that the5′-end of an antisense strand is linked to the 3′-end of a sense strand.Provided hairpin oligonucleotides are also referred to herein as“shRNA”.

Single-Stranded Oligonucleotides

In some embodiments, a single-stranded oligonucleotide utilized inaccordance with the present invention comprises a nucleotide sequencethat is substantially complementary to a “sense” nucleic acid encoding agene expression product, e.g., complementary to the coding strand of adouble-stranded cDNA molecule or complementary to an RNA sequence, e.g.,a pre-mRNA, mRNA, miRNA, or premiRNA. Provided single-strandedoligonucleotides include, but are not limited to, antisenseoligonucleotides and single-stranded RNAi agents. In some embodiments,the region of complementarity is less than about 30 nucleotides inlength. In some embodiments, the region of complementarity is at leastabout 15 nucleotides in length. In some embodiments, a provided singlestranded oligonucleotide is about 10 to about 25 nucleotides in length(e.g., about 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, or 24nucleotides in length). In some embodiments, a provided single strandedoligonucleotide is about 25 to about 30 nucleotides in length. In someembodiments, a provided single stranded oligonucleotide is about 15 toabout 29 nucleotides in length. In some embodiments, a provided singlestranded oligonucleotide is characterized as having less than 100%complementarity to a mRNA, RNA or DNA arc. In some embodiments, aprovided single-stranded oligonucleotide has a ZXY structure, such as isdescribed in International Application No. PCT/US2004/07070 filed onMar. 8, 2004.

In some embodiments, a utilized single-stranded oligonucleotide canhybridize to a complementary RNA, e.g., mRNA, pre-mRNA, and preventaccess of the translation machinery to the target RNA transcript,thereby preventing protein synthesis. In some embodiments, a providedsingle-stranded oligonucleotide can hybridize to a complementary RNA andthe RNA target can be subsequently cleaved by an enzyme such as RNasc H,thus preventing translation of target RNA. In some embodiments, aprovided single-stranded oligonucleotide modulates the expression of atarget gene via RISC mediated cleavage of the target sequence.

A “single-stranded RNAi agent” as used herein, is an RNAi agent which ismade up of a single molecule. In some embodiments, a single-strandedRNAi agent includes a duplexed region, formed by intra-strand pairing,e.g., it is or it includes a hairpin or pan-handle structure. In someembodiments, single-stranded RNAi agents are antisense with regard tothe target molecule. In some embodiments, single-stranded RNAi agentsare sufficiently long such that they are able to enter the RISC andparticipate in RISC mediated cleavage of a target mRNA. Exemplarysingle-stranded siRNAs (ss siRNAs) are known and are described, forexample, in U.S. Pat. Pub. No. 2006/0166901, the contents of which areherein incorporated by reference in its entirety.

In some embodiments, a single-stranded RNAi agent is at least about 12nucleotides in length. In some embodiments, a single-stranded RNAi agentis at least about 15 nucleotides in length. In some embodiments, asingle-stranded RNAi agent is at least about 20 nucleotides in length.In some embodiments, a single-stranded RNAi agent is at least about 25nucleotides in length. In some embodiments, a single-stranded RNAi agentis at least about 29 nucleotides in length. In some embodiments, asingle-stranded RNAi agent is at least about 30 nucleotides in length.In some embodiments, a single-stranded RNAi agent is at least about 35nucleotides in length. In some embodiments, a single-stranded RNAi agentis at least about 40 nucleotides in length. In some embodiments, asingle-stranded RNAi agent is at least about 50 nucleotides in length.

In some embodiments, a single-stranded RNAi agent is less than 200nucleotides in length. In some embodiments, a single-stranded RNAi agentis less than 100 nucleotides in length. In some embodiments, asingle-stranded RNAi agent is less than 60 nucleotides in length.

In some embodiments. a single-stranded RNAi agent is 5′ phosphorylated.In some embodiments, a singlestranded RNAi agent includes a phosphorylanalog at the 5′ prime terminus. In certain embodiments, asingle-stranded RNAi agent has length from about 15 to about 29nucleotides in length.

Single-stranded oligonucleotides, including those described and/oridentified as single stranded siRNAs, microRNAs or mirs which may beused as targets or may serve as a template for the design ofoligonucleotides of the invention are taught in, for example, Esau, etal. US Publication #20050261218 (U.S. Ser. No. 10/909,125) entitled“Oligonucleotides and compositions for use in modulation smallnon-coding RNAs” the entire contents of which are incorporated herein byreference.

The present invention encompasses research and/or diagnostic reagentsthat comprise single-stranded oligonucleotides. In some embodiments, asingle-stranded oligonucleotide utilized in accordance with the presentinvention is and/or acts as a primer. In some embodiments, primers areused in polymerase-based chain reactions (i.e., PCR) to amplify nucleicacids. These applications include any known variations of PCR, such asreverse transcription PCR (RT-PCR) and real-time PCR.

MicroRNAs

In some embodiments provided compositions comprise one or moreoligonucleotides that are or act as MicroRNAs.

MicroRNAs (miRNAs or mirs) are a highly conserved class of small RNAmolecules that are transcribed from DNA in the genomes of plants andanimals, but are not translated into protein. Pre-microRNAs areprocessed into miRNAs. Processed microRNAs are single stranded 17-25nucleotide (nt) RNA molecules that become incorporated into theRNA-induced silencing complex (RISC) and have been identified as keyregulators of development, cell proliferation, apoptosis anddifferentiation. They are believed to play a role in regulation of geneexpression by binding to the 3′-untranslated region of specific mRNAs.RISC mediates downregulation of gene expression through translationalinhibition, transcript cleavage, or both. RISC is also implicated intranscriptional silencing in the nucleus of a wide range of eukaryotes.

MicroRNAs have also been implicated in modulation of pathogens in hosts.For example, see Jopling, C. L., et al., Science (2005) vol. 309, pp1577-1581. Without wishing to be bound by theory, administration of amicroRNA, microRNA mimic, and/or anti microRNA oligonucleotide, leads tomodulation of pathogen viability, growth, development, and/orreplication. In some embodiments, a provided oligonucleotide is amicroRNA, microRNA mimic, and/or anti microRNA, wherein microRNA is ahost microRNA. The number of miRNA sequences identified to date is largeand growing, illustrative examples of which can be found, for example,in: “miRBase: microRIVA sequences, targets and gene nomenclature”Griffiths-Jones S, Grocock R J, van Dongen S, Bateman A, Enright A J.NAR, 2006, 34, Database Issue, D140-D144; “The microRNA Registry”Griffiths-Jones S. NAR, 2004, 32, Database Issue, D109-D111.Non-limiting examples of useful miRNA sequences are also provided in theaccompanying Appendix (C).

Ribozymes

In some embodiments, provided compositions include one or moreoligonucleotides that are or act as ribozymes.

Ribozymes are oligonucleotides having specific catalytic domains thatpossess endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA.1987 December; 84(24):8788-92; Forster and Symons, Cell. 1987 Apr. 24;49(2):211-20). At least six basic varieties of naturally occurringenzymatic RNAs are known presently. In general, enzymatic nucleic acidsact by first binding to a target RNA. Such binding occurs through thetarget binding portion of an enzymatic nucleic acid which is held inclose proximity to an enzymatic portion of the molecule that acts tocleave the target RNA. Thus, the enzymatic nucleic acid first recognizesand then binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

Methods of producing a ribozyme targeted to any target sequence areknown in the art. Ribozymes can be designed as described in, inter alia,Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO94/02595, each specifically incorporated herein by reference, andsynthesized to be tested in vitro and in vivo, as described therein.

Aptamers

In some embodiments, provided compositions include one or moreoligonucleotides that are or act as aptamers.

Aptamers are nucleic acid or peptide molecules that bind to a particularmolecule of interest with high affinity and specificity (Tuerk and Gold,Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)).DNA or RNA aptamers have been successfully produced which bind manydifferent entities from large proteins to small organic molecules. SeeEaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin.Struct. Biol. 9:324-9(1999), and Hermann and Patel, Science 287:820-5(2000). Aptamers can be RNA or DNA based. Generally, aptamers areengineered through repeated rounds of in vitro selection orequivalently, SELEX (systematic evolution of ligands by exponentialenrichment) to bind to various molecular targets such as smallmolecules, proteins, nucleic acids, and even cells, tissues andorganisms. The SELEX procedure is a protocol in which single strandedoligonucleotides are selected from vast libraries of sequences, based onbinding affinity at a target protein or other molecule (C. Tuerk, L.Gold, Science, 249 (1990), pp. 505-510; R. Green, A. D. Ellington, D. P.Bartel, J. W. Szostak, Methods Enzymol., 2 (1991), pp. 75-86; L. Gold,B. Polisky, O. Uhlenbeck, M. Yarus, Annu. Rev. Biochem., 64 (1995), pp.763-797). The SELEX procedure is usually initiated with an RNA or DNAlibrary consisting of some 10¹⁴-10¹⁵ random oligonucleotide sequences.In a fully randomised oligonucleotide library, each molecule willexhibit a unique tertiary structure which will be dependent on thenucleotide sequence of that molecule. The binding affinity of theoligonucleotide for the target protein will be determined by the fitbetween moieties on the surface of the oligonucleotide and epitopes onthe target protein. As a consequence of starting from a library of vastdiversity it is often possible to identify aptamers of nM or sub-nMaffinity for the target protein and with selectivity for that targetprotein over other proteins with a high degree of structural homology(K. W. Uphoff, S. D. Bell, A. D. Ellington, Curr. Opin. Struct. Biol., 6(1996), pp. 281-288). Using SELEX methodology RNA or DNA aptamers havebeen generated to many proteins, peptides and small molecules includingdopamine (C. Mannironi, A. Di Nardo, P. Fruscoloni, G. P.Tocchini-Valentini, Biochemistry, 36 (1997), pp. 9726-9734), substance P(D. Nieuwlandt, M. Wecker, Biochemistry, 34 (1995), pp. 5651-5659),subtilisin (H. Takeno, S. Yamamoto, T. Tanaka, Y. Sakano, Y. Kikuchi, J.Biochem., 125 (1999), pp. 1115-1119), platelet derived growth factor (L.S. Green, D. Jellinek, R. Jenison, A. Ostman, C-H. Heldin, N. JanjicBiochemistry, 35 (1996), pp. 14413-14424), vascular endothelial growthfactor (L. S. Green, D. Jellinek, C. Bell, L. A. Bebe, B. D. Feistner,S. C. Gill, F. M. Jucker, N. Janjic Chem. Biol., 2 (1995), pp. 683-695),thrombin (L. C. Bock, L. C. Griffen, J. A. Latham, E. H. Vermaas, J. J.Toole, Nature, 355 (1992), pp. 564-566), and L-selectin (D. O'Connell,A. Koenig, S. Jennings, B. Hicke, H. L. Han, T. Fitzwater, Y. F. Chang,N. Varki, D. Parma Proc. Natl. Acad. Sci. USA, 93 (1996), pp.5883-5887).

As reviewed in Dua et al. (2008) Recent Patents on DNA & Gene Sequences,2: 172-186 (incorporated herein by reference) in more detail, over theyears, a number of modified SELEX protocols have been developed.Non-limiting examples of modified SELEX methods are described in thefollowing publications: Counter SELEX (U.S. Pat. No. 5,580,737), Flowcell SELEX (WO9833941), Truncation SELEX (WO0056930), Blended SELEX(U.S. Pat. No. 5,683,867), Transcription free SELEX (US20026387620),Solution SELEX (U.S. Pat. No. 5,567,588), Chimeric SELEX (WO9604403),Tissue SELEX (US20026376474), Photo SELEX (U.S. Pat. No. 6,001,577),Toggle SELEX (US20077312325), Covalent SELEX/Chemi SELEX (U.S. Pat. No.5,763,595), Genomic SELEX (US20016261774), SELEX without purifiedprotein (WO0224954), CE-SELEX (WO03102212), Mirror-imageSELEX—Spiegelmers (EP1386972) and Laser SELEX, DeSELEX (WO07035532),each of which is incorporated herein by reference in its entirety.Stereo-defined oligonucleotides embraced by the present invention may beused in any one or more of the variations of the SELEX methods.

An aptamer can be prepared by any known method, including synthetic,recombinant, and purification methods, and can be used alone or incombination with other aptamers specific for the same target. Further,as described more fully herein, the term “aptamer” specifically includes“secondary aptamers” containing a consensus sequence derived fromcomparing two or more known aptamers to a given target. Aptamers possessseveral characteristics that make them attractive therapeutic agents.Both DNA and RNA aptamers have been shown to bind their targets withdissociation constants (Kd) in the low picomolar to low nanomolar range.Binding of an aptamer is a highly specific interaction that can evendiscriminate between related proteins that share common structuraldomains. Though the binding affinity of both aptamers and antibodies isin the same range, aptamers have many additional features whichoverpower their rival in many cases. Unlike antibodies, aptamers can betargeted even against non immunogenic targets. During synthesis they canbe easily subjected to a chemical modification that improves theirstability and pharmacokinetics. They display nil to negligible levels ofimmunogenicity at therapeutic doses due to their resemblance toendogenous molecules.

In some embodiments, oligonucleotides utilized in accordance with thepresent invention are useful as anti-pathogenic agents, for example,antiviral agents, antibacterial agents, antifungal agents, and so on.Suitable targets for a variety of infectious agents such as viruses andbacteria have been reported. See, for example, U.S. Pat. Nos. 5,726,017,5,654,151, 5,496,938, 5,503,978, 5,587,468, 5,527,894, US2005233317,U.S. Pat. No. 5,496,938, WO08 066231, JP2002165594, WO02081494, U.S.Pat. No. 5,861,501, WO9720072, U.S. Pat. Nos. 5,475,096, 6,569,630,CN101109013, and WO03106476, each of which is incorporated herein byreference in its entirety.

In some embodiments, oligonucleotides utilized in accordance with thepresent invention are or act as anticancer agents. Any known cancer- ortumor-associated factors, such as proteins involved in the regulation ofcell division or proliferation, cell cycle progression, apoptosis, cellmigration, DNA repair, etc. including structural proteins and signaltransduction molecules, may be targeted by an aptamer. Examples include,without limitation, See, for example, AU Patent 775412, U.S. Pat. No.6,232,071, WO 08/028534, U.S. Pat. No. 6,933,114, WO 04/081574, AUPatent 242462, U.S. Pat. Nos. 6,995,249, 5,668,264, 6,699,843, WO04/094614, and U.S. Pat. No. 5,846,713, each of which is incorporatedherein by reference in its entirety.

In some embodiments, oligonucleotides utilized in accordance with thepresent invention are or act as antiangiogenic agents. Non-limitingexamples of antiangiogenic targets include VEGF and associatesreceptors, as well as extracellular matrix or adhesion molecules. See,for example, U.S. Pat. No. 6,051,698, US 2003175703, WO 95/21853 andU.S. Pat. No. 7,094,535, each of which is incorporated herein byreference in its entirety.

In some embodiments, oligonucleotides utilized in accordance with thepresent invention are or act as anticoagulant agents. Much is knownabout blood coagulation factors and their function as well as proteinsregulation such process. Aptamers are useful in targeting one or more ofsuch factors in treating conditions such as cardiovascular diseases andblood coagulation disorders. See for example, WO 06/033854, U.S. Pat.No. 5,543,293, WO 07/025049, U.S. Pat. No. 6,774,118, WO 07/140000, eachof which is incorporated herein by reference in its entirety.

In some embodiments, oligonucleotides utilized in accordance with thepresent invention are or act as immunomodulatory agents. Aptamers havebeen generated to target immunomodulatory molecules involved inautoimmune disorders. The present invention may be useful for targetingmolecules expressed on immune cells, such as T cells and antigenpresenting cells (APCs). See for example, U.S. Pat. No. 5,869,641, WO01/09160, each of which is incorporated herein by reference in itsentirety. In some embodiments, molecules involved in the complementsystem such as C1, C4, C2, C3 and C5 may be targeted by an aptamer. Thecomplement system has been implicated in numerous renal,rheumatological, neurological, dermatological, hematological, allergic,infectious, and other diseases. See for example, WO 97/28178 and WO07/103549, each of which is incorporated herein by reference in itsentirety. Other immune-related targets include, without limitation,IL-12, IL-23 and IL-28 (see, for example, WO 07/035922, which isincorporated herein by reference), IgE (see, for example, WO 05/113813,which is incorporated herein by reference), Sp-1 and Sp1-, CD28, IL-2,GMCSF (see, for example, U.S. Pat. No. 6,994,959, which is incorporatedherein by reference), SDF-1, CXCR4 (see, for example, WO 08/009437,which is incorporated herein by reference), IL-6, IL-12, IFN gamma (see,for example, U.S. Pat. No. 6,589,940 and US2003125279, each of which isincorporated herein by reference), and TLR. Stereo-defined aptamers mayelicit improved efficacy against such diseases.

In some embodiments, oligonucleotides utilized in accordance with thepresent invention are or act as anti-inflammatory agents. Inflammatorydisease such as acute respiratory distress syndrome (ARDS), septicshock, pulmonary emphysema, cystic fibrosis, rheumatoid arthritis andchronic bronchitis, have neutrophil elastase involved in theirpathogenesis. Human elastate is therefore a target for treating suchdisorders using an aptamer that regulates inflammation. Other suitabletargets include, without limitation, phospholipase A2, such asnon-pancreatic secretory PLA2 (see, for example, WO 96/27604, which isincorporated by reference), selectins, E-, P- and L- (see, for example,U.S. Pat. No. 5,780,228, which is incorporated by reference), threehomologous C-type lectins, other cell adhesion molecules expressed incells such as leukocytes, endothelial cells and platelets; MCP-1 (see,for example, WO 07/093409, which is incorporated by reference), NF-kappaB and NF-IL6 (see, for example, WO 00/24404, which is incorporated byreference). Stereo-defined aptamers may elicit improved efficacy againstsuch diseases.

In some embodiments, oligonucleotides utilized in accordance with thepresent invention are useful for treating certain brain diseases,including but are not limited to: Transmissible spongiformencephalopathies (TSEs) and Alzheimer's disease. Known targets aredescribed in publications including WO 2006/138676 and DE19916417, andWO 08/008884, each of which is incorporated herein by reference.Stereo-defined aptamers may elicit improved efficacy against suchdiseases.

Decoy Oligonucleotides

In some embodiments, provided compositions include one or moreoligonucleotides that are or act as decoy oligonucleotides.

Because transcription factors recognize their relatively short bindingsequences, even in the absence of surrounding genomic DNA, shortoligonucleotides bearing the consensus binding sequence of a specifictranscription factor can be used as tools for manipulating geneexpression in living cells. This strategy involves the intracellulardelivery of such “decoy oligonucleotides”, which are then recognized andbound by the target factor. Occupation of the transcription factor'sDNA-binding site by the decoy renders the transcription factor incapableof subsequently binding to the promoter regions of target genes. Decoyscan be used as therapeutic agents, either to inhibit the expression ofgenes that are activated by a transcription factor, or to up-regulategenes that are suppressed by the binding of a transcription factor.Examples of the utilization of decoy oligonucleotides can be found inMann et al., J. Clin. Invest., 2000, 106: 1071-1075, which is expresslyincorporated by reference herein, in its entirety.

miRNA Mimics

In some embodiments, provided compositions include one or moreoligonucleotides that are or act as miRNA mimics.

miRNA mimics represent a class of molecules that can be used to imitatethe gene modulating activity of one or more miRNAs. Thus, the term“microRNA mimic” refers to synthetic non-coding RNAs (i.e. the miRNA isnot obtained by purification from a source of the endogenous miRNA) thatare capable of entering the RNAi pathway and regulating gene expression.miRNA mimics can be designed as mature molecules (e.g. single stranded)or mimic precursors (e.g., pri- or pre-miRNAs).

In some embodiments, miRNA mimics are double stranded molecules (e.g.,with a duplex region of between about 16 and about 31 nucleotides inlength) and contain one or more sequences that have identity with themature strand of a given miRNA.

In some embodiments, an miRNA mimic comprises a duplex region of betweenabout 16 and about 31 nucleotides. In some embodiments, provided miRNAmimic may contain one or more of the following chemical modificationpatterns: the sense strand contains 2′-O-methyl modifications ofnucleotides 1 and 2 (counting from the 5′ end of the senseoligonucleotide), and all of the Cs and Us; the antisense strandmodifications can comprise 2′ F modification of all of the Cs and Us,phosphorylation of the 5′ end of the oligonucleotide, and stabilizedinternucleotide linkages associated with a 2 nucleotide 3′ overhang.

Supermirs

In some embodiments, provided compositions include one or moreoligonucleotides that are or act as supermirs.

A supermir refers to an oligonucleotide, e.g., single stranded, doublestranded or partially double stranded, which has a nucleotide sequencethat is substantially identical to an miRNA and that is antisense withrespect to its target. This term includes oligonucleotides whichcomprise at least one non-naturally-occurring portion which functionssimilarly. In some embodiments, the supermir does not include a sensestrand. In some embodiments, the supermir does not self-hybridize to asignificant extent. In some embodiments, a supermir has a secondarystructure but is substantially single-stranded under physiologicalconditions. A supermir that is substantially single-stranded issingle-stranded to the extent that less than about 50% (e.g., less thanabout 40%, 30%, 20%, 10%, or 5%) of the supermir is duplexed withitself. A supermir can include a hairpin segment, e.g., sequence,preferably at the 3′ end can self hybridize and form a duplex region,e.g., a duplex region of at least 1, 2, 3, or 4 and preferably less than8, 7, 6, or 5 nucleotides, e.g., 5 nucleotides. The duplexed region canbe connected by a linker, e.g., a nucleotide linker, e.g., 3, 4, 5, or 6dTs, e.g., modified dTs. In some embodiments, a supermir is duplexedwith a shorter oligonucleotide, e.g., of 5, 6, 7, 8, 9, or 10nucleotides in length, e.g., at one or both of the 3′ and 5′ end or atone end and in the non-terminal or middle of the supermir.

Antimers or miRNA Inhibitors

In some embodiments, provided compositions include one or moreoligonucleotides that are or act as antimers or miRNA inhibitors.

The terms “antimir” “microRNA inhibitor” or “miR inhibitor” aresynonymous and refer to oligonucleotides or modified oligonucleotidesthat interfere with the activity of specific miRNAs. Inhibitors canadopt a variety of configurations including single stranded, doublestranded (RNA/RNA or RNA/DNA duplexes), and hairpin designs. In someembodiments, microRNA inhibitors comprise one or more sequences orportions of sequences that are complementary or partially complementarywith the mature strand (or strands) of the miRNA to be targeted. In someembodiments, miRNA inhibitors comprise additional sequences located 5′and 3′ to the sequence that is the reverse complement of the maturemiRNA. Additional sequences can be the reverse complements of thesequences that are adjacent to the mature miRNA in the primiRNA fromwhich the mature miRNA is derived, or additional sequences can bearbitrary sequences (having a mixture of A, G, C, U, or dT). In someembodiments, one or both of the additional sequences are arbitrarysequences capable of forming hairpins. Thus, in some embodiments, thesequence that is the reverse complement of the miRNA is flanked on the5′ side and on the 3′ side by hairpin structures. In some embodiments,microRNA inhibitors are double stranded. In some embodiments, microRNAinhibitors are double stranded and include mismatches betweennucleotides on opposite strands. In some embodiments, microRNAinhibitors are linked to conjugate moieties in order to facilitateuptake of the inhibitor into a cell.

MicroRNA inhibitors, including hairpin miRNA inhibitors, are describedin detail in Vermeulen et al., “Double-Stranded Regions Are EssentialDesign Components Of Potent Inhibitors of RISC Function,” RNA 13:723-730 (2007) and in W02007/095387 and WO 2008/036825 each of which isincorporated herein by reference in its entirety.

An exemplary application of miRNA-based therapy is described in Pan etal. (2007) World J Gastroenterol 13(33): 4431-36, “New therapeuticopportunities for Hepatitis C based on small RNA,” the contents of whichare incorporated by reference. Briefly, the authors describe 22nucleotide mature miR-122, derived from a noncoding polyadenylated RNAtranscript of the_hcr gene, which is a liver-specific developmentalregulator. Because miR-122 is a liver specific miRNA that is involved inHCV viral replication, silencing of miR-122 can be useful for thetreatment of HCV.

Antagomirs

In some embodiments, provided compositions include one or moreoligonucleotides that are or act as antagomirs.

Antagomirs are RNA-like oligonucleotides that harbor variousmodifications for RNAse protection and pharmacologic properties, such asenhanced tissue and cellular uptake. They differ from normal RNA by, forexample, complete 2′-0-methylation of sugar, phosphorothioate intersugarlinkage and, for example, a cholesterol-moiety at 3′-end. In someembodiments, an antagomir comprises a 2′-O-methylmodification at allnucleotides, a cholesterol moiety at 3′-end, two phosphorothioateintersugar linkages at the first two positions at the 5′-end and fourphosphorothioate linkages at the 3′-end of the molecule. Antagomirs canbe used to efficiently silence endogenous miRNAs by forming duplexescomprising the antagomir and endogenous miRNA, thereby preventingmiRNA-induced gene silencing. An example of antagomir-mediated miRNAsilencing is the silencing of miR-122, described in Krutzfeldt et al,Nature, 2005, 438: 685-689, which is incorporated by reference herein inits entirety.

Modified Single Oligonucleotide Variations

Typically, it is perceived that two components are required to activateRNAi machinery—the double-stranded nucleic acid motif, which is requiredfor recognition by RNAi-associated proteins and the guide strand whichserves as the mRNA binding co-factor in the RISC's Argonaute catalyticprotein. More recently, a novel type of RNAi molecules composed of asingle, short (25-28 nt) oligo capable of self-dimerizing into apartially complementary duplex have been developed. These molecules weredemonstrated to efficiently activate the RISC and to produce target mRNAsilencing comparable to that obtained with potent conventional RNAimolecules. See: Lapierre et al., “Potent and systematic RNAi mediatedsilencing with single oligonucleotide compounds.” RNA 2011; 17:00, thecontents of which are hereby incorporated by reference. See also: WO2010/090762 “RNA DUPLEXES WITH SINGLE STRANDED PHOSPHOROTHIOATENUCLEOTIDE REGIONS FOR ADDITIONAL FUNCTIONALITY” (PCT/US2010/00348), thecontents of which are hereby incorporated by reference.

Thus, the present invention includes RNAi constructs containing singlestranded regions of phosphorothioate modified nucleotides, and the usesof such constructs in gene silencing.

In some embodiments, the invention utilizes isolated double strandednucleic acid molecules including a guide strand and a passenger strand,wherein the passenger strand is connected through a cleavable linker toa single stranded region of at least eight phosphorothioate modifiednucleotides. In some embodiments, the invention is an isolated doublestranded nucleic acid molecule having a guide strand and a passengerstrand, wherein at least one of the guide strand and passenger strand isconnected through a cleavable linker to a single stranded region of atleast six phosphorothioate modified nucleotides.

In some embodiments, the invention utilizes an isolated double strandednucleic acid molecule having a guide strand and a passenger strand,wherein at least one of the guide strand and passenger strand isconnected through a cleavable linker to a single stranded region of atleast three phosphorothioate modified nucleotides. In some embodiments,the double stranded nucleic acid molecule includes at least one of thefollowing properties. The passenger strand may be 8-18 nucleotides inlength. The nucleic acid may have at least one 2′ O methyl or 2′ fluoromodification. The cleavable linkage may be other than a nucleotidiclinkage. The nucleic acid may include a lipophilic group. The guidestrand may be 16-18 nucleotides or 26-28 nucleotides in length. In someembodiments the single stranded region is connected to the guide strand.

In some embodiments, the cleavable linker includes one or moreunmodified nucleotides. In other embodiments, the cleavable linker is aphosphodiester bond. In certain embodiments, the cleavable linker isS—S. In some embodiments the cleavable linker is DNA or RNA. The singlestranded region of at least eight phosphorothioate modified nucleotidescan be at either the 3′ or 5′ end of the passenger strand. In someembodiments, the single stranded region of at least eightphosphorothioate modified nucleotides is DNA, while in other embodimentsit is RNA.

In some embodiments the double stranded region of the nucleic acidmolecule is a perfect duplex. In other embodiments the double strandedregion contains at least one bulge region. In some embodiments thepassenger strand comprises a nick within the double stranded region ofthe molecule. The double stranded region may contain at least onenucleotide that is phosphorothioate modified.

Nucleic acid molecules utilized in accordance with the invention may bechemically modified. In certain embodiments the chemical modification is2′Omethyl and/or 2′Fluoro. In some embodiments more than one chemicalmodification is present in the same molecule. In some embodimentschemical modification increases stability, increases evasion of immuneregulation, and/or prevents off-target gene silencing. Chemicalmodification can be present on the passenger strand and/or the guidestrand. In some embodiments the single stranded region of at least eightphosphorothioate modified nucleotides is cleaved from the doublestranded region of the nucleic acid molecule in a cell. In someembodiments the single stranded region of at least eightphosphorothioate modified nucleotides has complementarity to a mammaliangene. In certain embodiments the single stranded region of at leasteight phosphorothioate modified nucleotides functions as an antisensemolecule. The double stranded region may be at least 19 nucleotideslong. In some embodiments the single stranded region is at least 12nucleotides long.

In some embodiments, the invention utilizes bifunctional nucleic acidmolecule including a double stranded region that functions in RNAinterference and a single stranded region that functions in antisense,wherein the double stranded region comprises a guide strand and apassenger strand, and wherein the double stranded region and the singlestranded region are connected through a cleavable linker. In someembodiments, the cleavable linker includes one or more unmodifiednucleotides. In other embodiments, the cleavable linker is aphosphodiester bond. In certain embodiments, the cleavable linker isS—S. In some embodiments the cleavable linker is DNA or RNA.

In some embodiments, the invention relates to methods for inhibiting theexpression of a target gene in a mammalian cell, including contactingthe mammalian cell with an isolated double stranded nucleic acidmolecule comprising a guide strand and a passenger strand, wherein thepassenger strand is connected through a cleavable linker to a singlestranded region of at least eight phosphorothioate modified nucleotides.In some embodiments, the cleavable linker includes one or moreunmodified nucleotides. In other embodiments, the cleavable linker is aphosphodiester bond. In certain embodiments, the cleavable linker isS—S. In some embodiments the cleavable linker is DNA or RNA.

In some embodiments, the single stranded region comprises at least eightphosphorothioate modified nucleotides and can be at either the 3′ or 5′end of the passenger strand. In some embodiments, the single strandedregion of at least eight phosphorothioate modified nucleotides is DNA,while in other embodiments it is RNA. In some embodiments the doublestranded region of the nucleic acid molecule is a perfect duplex. Inother embodiments the double stranded region contains at least one bulgeregion. In some embodiments the passenger strand comprises a nick withinthe double stranded region of the molecule. The double stranded regionmay contain at least one nucleotide that is phosphorothioate modified.

Nucleic acid molecules utilized in accordance with the invention may bechemically modified. In certain embodiments the chemical modification is2′Omethyl and/or 2′Fluoro. In some embodiments more than one chemicalmodification is present in the same molecule. In some embodimentschemical modification increases stability, increases evasion of immuneregulation, and/or prevents off-target gene silencing. Chemicalmodification can be present on the passenger strand and/or the guidestrand.

In some embodiments the single stranded region of at least eightphosphorothioate modified nucleotides is cleaved from the doublestranded region of the nucleic acid molecule in a cell. In someembodiments the single stranded region of at least eightphosphorothioate modified nucleotides has complementarity to a mammaliangene. In certain embodiments the single stranded region of at leasteight phosphorothioate modified nucleotides functions as an antisensemolecule. The double stranded region may be at least 19 nucleotideslong. In some embodiments the single stranded region is at least 12nucleotides long.

In some embodiments, the invention relates to methods for inhibiting theexpression of a target gene in a mammalian cell, comprising contactingthe mammalian cell with an isolated double stranded nucleic acidmolecule comprising a guide strand and a passenger strand, wherein theguide strand is connected through a cleavable linker to a singlestranded region of at least eight phosphorothioate modified nucleotides.In some embodiments, the cleavable linker includes one or moreunmodified nucleotides. In other embodiments, the cleavable linker is aphosphodiester bond. In certain embodiments, the cleavable linker isS—S. In some embodiments the cleavable linker is DNA or RNA.

In some embodiments, the invention relate to methods for inhibiting theexpression of a target gene in a mammalian cell, including contactingthe mammalian cell with a bifunctional nucleic acid molecule including adouble stranded region that functions in RNA interference and a singlestranded region that functions in antisense, wherein the double strandedregion includes a guide strand and a passenger strand, and wherein thedouble stranded region and the single stranded region are connectedthrough a cleavable linker. In other aspects a method for inhibiting theexpression of a target gene in a mammalian cell is provided. The methodinvolves contacting the mammalian cell with any of the isolated doublestranded nucleic acid molecules described herein.

In some embodiments, isolated double stranded nucleic acid moleculesutilized in accordance with the present invention include a chemicalmodification that increases stability. In some embodiments, isolateddouble stranded nucleic acid molecules include a chemical modificationthat increases evasion of immune regulation. In some embodiments, theisolated double stranded nucleic acid molecules include a chemicalmodification that prevents off-target gene silencing.

In some embodiments, as discussed herein, utilized oligonucleotides aresingle oligonucleotide molecules capable of self-dimerizing into apartially complementary duplex. In some embodiments, such singleoligonucleotides are about 23-30 nucleotides in length, e.g., 23, 24,25, 26, 27, 28, 29, and 30. In some embodiments, single oligonucleotidescapable of self-dimerizing into a partially complementary duplex containabout 14-18 nucleotide mRNA targeting region, e.g., 14, 15, 16, 17, and18. In some embodiments, single oligonucleotides capable ofself-dimerizing into a partially complementary duplex contain anadditional 7-11, e.g., 7, 8, 9, 10, and 11 nucleotides to enableself-dimerization into a partially complementary duplex. In someembodiments, single oligonucleotides capable of self-dimerizing into apartially complementary duplex can efficiently enter and activate theRNA-induced silencing complex (RISC).

Ul Adaptors

In some embodiments, provided compositions include one or moreoligonucleotides that are or act as U1 adaptors.

Ul adaptors inhibit polyA sites and are bifunctional oligonucleotideswith a target domain complementary to a site in the target gene'sterminal exon and a ‘Ul domain’ that binds to the Ul smaller nuclear RNAcomponent of the Ul snRNP. See for example, Int. Pat. App. Pub. No.W02008/121963 and Goraczniak, et al., 2008, Nature Biotechnology, 27(3),257-263, each of which is incorporated by reference herein, in itsentirety. Ul snRNP is a ribonucleoprotein complex that functionsprimarily to direct early steps in spliceosome formation by binding tothe pre-mRNA exon-intron boundary, Brown and Simpson, 1998, Annu RevPlant Physiol Plant MoI Biol 49:77-95.

In some embodiments, a utilized oligonucleotide is a Ul adaptor, whereinthe oligonucleotide comprises at least one annealing domain (targetingdomain) linked to at least one effector domain (U1 domain), wherein theannealing domain hybridizes to a target gene sequence and the effectordomain hybridizes to the Ul snRNA of Ul snRNP. In some embodiments, theUl adaptor comprises one annealing domain. In some embodiments, the Uladaptor comprises one effector domain.

Without wishing to be bound by theory, the annealing domain willtypically be from about 10 to about 50 nucleotides in length, moretypically from about 10 to about 30 nucleotides or about 10 to about 20nucleotides. In some embodiments, the annealing, domain is 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides in length. Theannealing domain may be at least 75%, at .least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, or at least about 100%complementary to the target gene. In some embodiments, the annealingdomain hybridizes with a target site within the Y terminal exon of apre-MRNA, which includes the terminal coding region and the 3′UTR andpolyadenylation signal sequences (e.g., through the polyadenylationsite). In some embodiments, the target sequence is within about500′basepair, about 250 basepair, about 100 basepair, or about 50⁻basepair of the poly (A) signal sequence of the, pre-mRNA. In someembodiments, the annealing domain comprises 1, 2, 3, or 4, mismatcheswith the target gene sequence.

In some embodiments, the effector domain is from about 8 nucleotides toabout 30 nucleotides in length. In some embodiments, the effector domainis from about 10 nucleotides to about 20 nucleotides in length. In someembodiments, the effector domain is from about 10 nucleotides to about15 nucleotides in length. The Ul domain can hybridize with UI snRNA,particularly the 5′ end and more specifically nucleotides 2-11. In someembodiments, the Ul domain, is perfectly complementary to nucleotides2-11 of endogenous U1 snRNA. In some embodiments, the U1 domaincomprises a nucleotide sequence selected from the group consisting ofSEQ. ID NO: 2299 (5′-GCCAGGUAAGUAU-3′), SEQ. 1D NO:2300(5′-CCAGGUAAGUAU-3′). SEQ ID NO: 2301 (5′-CAGGUAAGUAU-3′), SEQ IDNO: 2302 (5′-CAGGUAAGU-3′), SEQ ID NO: 2303 (5′-CAGGUAAG-3′), and. SEQID NO: 2304 (5′-CAGGUAA 3′). In some embodiments; the III domaincomprises a nucleotide sequence, SEQ ID NO: 2305 (5′-CAGGUAAGUA-3′).Without wishing to be bound by theory, increasing the length of the U1domain to include basepairing into stem 1 and/or basepairing to position1 of U1 snRNA improves th.e U1 adaptor's affinity to Ul snRNA.

The annealing and effector domains of the Ul adaptor can be linked suchthat the effector domain is at the 5′ end and/or 3′ end of the annealingdomain. The two domains can be linked by such that the 3′ end of onedomain is linked to 5′ end of the other domain, or 3′ end of one domainis linked to 3′ end of the other domain, or 5′ end of one domain islinked to 5′ end of the other domain. The annealing and effector domainscan be linked directly to each other or by a nucleotide based ornon-nucleotide based linker. In some embodiments, a linker is nucleotidebase and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15, up to 20, orup to 25 nucleotides.

In some embodiments, a linker between an annealing domain and aneffector domain is multivalent, e.g., trivalent, tetravalent.pentavalent. Without wishing to be bound by theory, a multivalent linkercan be used to .link together a single annealing domain with a pluralityof adaptor domains.

In some embodiments, a Ul adaptor comprises any oligonucleotidemodification described herein. Exemplary such modifications includethose that increase annealing affinity, specificity, bioavailability inthe cell and organism, cellular and/or nuclear transport, stability,and/or resistance to degradation. In some embodiments, the Ul adaptorcan be administered in combination with at least one other RNAi agent.

RNA Activators

In some embodiments, provided compositions include one or moreoligonucleotides that are or act as RNA activators.

Recent studies have found that dsRNA can also activate gene expression,a mechanism that has been termed “small RNA-induced gene activation” orRNAa. See for example Li, L. C. et al. Proc Nati Auld Sci USA. (2006),103(46): 17337-42 and Li L. C. (2008). “Small RNAMediated GeneActivation”. RNA and the Regulation of Gene Expression: A Hidden Layerof Complexity. Caister Academic Press. ISBN 978-1-904455-25-7. It hasbeen shown that dsRNAs targeting gene promoters induce potenttranscriptional activation of associated genes. Endogenous miRNA thatcause RNAa has also been found in humans. Check E. Nature (2007). 448(7156): 855-858.

Another observation is that gene activation by RNAa is long-lasting.Induction of gene expression has been seen to last for over ten days.The prolonged effect of RNAa could be attributed to epigenetic changesat dsRNA target sites.

In some embodiments, a provided oligonucleotide is an RNA activator,wherein a provided oligonucleotide increases the expression of a gene.In some embodiments, increased gene expression inhibits viability,growth development, and/or reproduction.

Non-Coding RNAs (ncRNAs)

The present invention encompasses non-coding RNAs, such as longnon-coding RNAs (lncRNAs) and short non-coding RNAs (sncRNAs).

Accordingly, provided oligonucleotide compositions of the presentinvention may be useful for modulating disease-associated longnon-coding RNA. In some embodiments, oligonuclotides synthesizedaccording to the methods provided herein are used to specifically blockthe binding of transcriptional repressor complexes to target lncRNAregions, thereby inducing the expression of the associated target gene.In some embodiments, the transcriptional repressor complex is asilencing factor. In some embodiments, the transcriptional repressorcomplex has Histone methyltransferase activity. In some embodiments, thetranscriptional repressor complex methylates histone H3. In someembodiments, the transcriptional repressor complex is PRC2 (PolycombRepressive Complex 2).

Certain PRC2-associated lncRNAs have been reported to be potentialtherapeutic targets and/or biomarkers (Zhao et al., 2010. “Genome-wideIdentification of Polycomb-Associated RNAs by RIP-seq” Molecular Cell40: 939-53). Overexpression of PCR2 proteins have been linked to varioustypes of cancer, including metastatic prostate and breast cancer, andcancers of the colon, breast, and liver. Pharmacological inhibition ofPRC2-mediated gene repression was found to induce apoptosis in severalcancer cell lines in vitro, but not in various types of normal cells.Induction of apoptosis in this system is dependent on reactivation ofgenes that had been repressed by PRC2. There is also evidence thatPRC2-mediated gene repression may be linked to the maintenance of thestem-cell properties of cancer stem cells. These results suggest that atleast in some cases, inhibition of PRC2-mediated generepression-including via targeting lncRNAs that recruit PRC2 to criticalgenes—is a potential strategy for treating various types of cancer.Non-limiting sequences of nucleic acids, which can be prepared inaccordance with the methods provided herein, can be found in, forexample, International Patent Publication WO 2012/065143 entitled“Polycomb-associated non-coding RNAs” (PCT/US2011/60493), the contentsof which are incorporated by reference herein.

Provided oligonucleotide compositions of the present invention may be oract as small non-coding RNAs, such as piwi-interacting RNAs (piRNAs).The piRNAs represent the largest class of small non-coding RNA moleculesthat is expressed in animal cells and are found in clusters throughoutthe genome. piRNAs are known to form RNA-protein complexes throughinteractions with piwi proteins. piRNA complexes have been implicated inboth epigenetic and post-transcriptional gene silencing ofretrotransposons and other genetic elements in germ cells, includingspermatogenesis. Typically piRNAs are 26-31 nucleotides in length, and,as compared to typical miRNAs, they lack sequence conservation andexhibit higher complexity. In some embodiments, oligonucleotidecompositions utilized in accordance with the present invention comprise5′ uridine. In some embodiments, oligonucleotide compositions utilizedin accordance with the present invention comprise 5′ monophosphate and a3′ modification that acts to block either the 2′ or 3′ oxygen. In someembodiments, the 3′ modification is a 2′-O-methylation.

Triplex Forming Oligonucleotides

In some embodiments, provided compositions include one or moreoligonucleotides that are or act as triplex-forming oligonucleotides.

Recent studies have shown that triplex forming oligonucleotides (TFO)can be designed which can recognize and bind topolypurine/polypyrimidine regions in double-stranded helical DNA in asequence-specific manner. These recognition rules are outline by MaherIII, L. J., et al., Science (1989) vol. 245, pp 725-730; Moser, H. E.,et al., Science (1987) vol. 238, pp 645-630; Beal, P. A., et al.,Science (1992) vol. 251, pp 1360-1363; Conney, M., et al., Science(1988) vol. 241, pp 456-459 and Hogan, M. E., et al., EP Publication375408. Modification of the oligonucleotides, such as the introductionof intercalators and intersugar linkage substitutions, and optimizationof binding conditions (pH and cation concentration) have aided in.overcoming inherent obstacles to TFO activity such as charge repulsionand instability, and it was recently shown that oligonucleotides can betargeted to specific sequences (for a recent review sec Seidman andGlazer, Clin Invest 2003 2:487-94). In generaL the triplex-formingoligonucleotide has the sequence correspondence:

oligo 3′-A G G T duplex 5′-A G C T duplex 3′-T C G A

However, it has been shown that the A-AT and G-GC triplets have thegreatest triple helical stability (Reither and Jeltsch, BMC Biochem,2002, Sep. 12, Epub). The same authors have demonstrated that TFOsdesigned according to the A-AT and G-GC rule do not form nonspecifictriplexes, indicating that the triplex formation is indeed sequencespecific. Thus for any given sequence a triplex forming sequence can bedevised. In some embodiments, triplex-forming oligonucleotides are atleast about 15, about 25, or about 30 or more nucleotides in length. Insome embodiments, triplex-forming oligonucleotides are up to about 50 orabout 100 nucleotides in length.

Formation of the triple helical structure with the target DNA inducessteric and functional changes, blocking transcription initiation andelongation, allowing the introduction of desired sequence changes in theendogenous DNA and resulting in the specific down-regulation of geneexpression. Examples of such suppression of gene expression in cellstreated with TFOs include knockout of episomal supFGI and endogenousHPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999; 27:1176-81, and Puri, et al, J Biol Chem, 2001; 276:28991-98), and thesequence- and target specific downregulation of expression of the Ets2transcription factor, important in prostate cancer etiology (Carbone, etal, Nucl Acid Res. 2003; 31:833-43), and the pro-inflammatory ICAM-Igene (Besch et al, J Biol Chem, 2002; 277:32473-79). In addition,Vuyisich and Beal have recently shown that sequence specific TFOs canbind to dsRNA, inhibiting activity dsRNA-dependent enzymes such asRNA-dependent kinases (Vuyisich and Beal, Nuc, Acids Res 2000;28:2369-74).

Additionally, TF0s designed according to the abovementioned principlescan induce directed mutagenesis capable of effecting DNA repair, thusproviding both down-regulation and up-regulation of expression ofendogenous genes (Seidman and Glazer, J Invest 2003; 112:487-94).Detailed description of the design, synthesis and administration ofeffective TFOs can be found. S. Pat. App. Nos. 2003 017068 and 20030096980 to Froehier et at and 2002 012821/8 and 2002 0123476 to Emanudeet aE, and U.S. Pat. No. 5,721,138 to Lawn, the contents of which areherein incorporated in their entireties.

Conjugates/Linkers

The invention also contemplates that utilized oligonucleotides, in someembodiments, are optimized for cellular uptake. In any utilizedoligonucleotides embraced by the present invention, guide and/orpassenger strands may be attached to a conjugate. In some embodimentsthe conjugate is hydrophobic. The hydrophobic conjugate can be a smallmolecule with a partition coefficient that is higher than 10. Theconjugate can be a sterol-type molecule such as cholesterol, or amolecule with an increased length polycarbon chain attached to C17, andthe presence of a conjugate can influence the ability of an RNA moleculeto be taken into a cell with or without a lipid transfection reagent.The conjugate can be attached to the passenger or guide strand through ahydrophobic linker. In some embodiments, a hydrophobic linker is 5-12Cin length, and/or is hydroxypyrrolidine-based. In some embodiments, ahydrophobic conjugate is attached to the passenger strand and the CUresidues of either the passenger and/or guide strand are modified. Insome embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%or 95% of the CU residues on the passenger strand and/or the guidestrand are modified. In some aspects, molecules associated with theinvention are self-delivering (sd). As used herein, “self-delivery”refers to the ability of a molecule to be delivered into a cell withoutthe need for an additional delivery vehicle such as a transfectionreagent. Aspects of the invention relate to selecting molecules for usein RNAi.

In any of the embodiments embraced herein, utilized oligonucleotides maybe associated with a hydrophobic moiety for targeting and/or delivery ofthe molecule to a cell. In some embodiments, the hydrophobic moiety isassociated with the nucleic acid molecule through a linker. In someembodiments, the association is through non-covalent interactions. Inother some, the association is through a covalent bond. Any linker knownin the art may be used to associate the nucleic acid with thehydrophobic moiety. Linkers known in the art are described in publishedinternational PCT applications, WO 92/03464, WO 95/23162, WO2008/021157, WO 2009/021157, WO 2009/134487, WO 2009/126933, U.S. PatentApplication Publication 2005/0107325, U.S. Pat. Nos. 5,414,077,5,419,966, 5,512,667, 5,646,126, and 5,652,359, which are incorporatedherein by reference. The linker may be as simple as a covalent bond to amulti-atom linker. The linker may be cyclic or acyclic. The linker maybe optionally substituted. In some embodiments, the linker is capable ofbeing cleaved from the nucleic acid. In certain embodiments, the linkeris capable of being hydrolyzed under physiological conditions. In someembodiments, the linker is capable of being cleaved by an enzyme (e.g.,an esterase or phosphodiesterase). In some embodiments, the linkercomprises a spacer element to separate the nucleic acid from thehydrophobic moiety. The spacer element may include one to thirty carbonor heteroatoms. In certain embodiments, the linker and/or spacer elementcomprises protonatable functional groups. Such protonatable functionalgroups may promote the endosomal escape of the nucleic acid molecule.The protonatable functional groups may also aid in the delivery of thenucleic acid to a cell, for example, neutralizing the overall charge ofthe molecule. In some embodiments, the linker and/or spacer element isbiologically inert (that is, it does not impart biological activity orfunction to the resulting nucleic acid molecule).

The hydrophobic molecule may be connected to the polynucleotide by alinker moiety. Optionally the linker moiety is a non-nucleotidic linkermoiety. Non-nucleotidic linkers are e.g. abasic residues (dSpacer),oligoethyleneglycol, such as triethyleneglycol (spacer 9) orhexaethyleneglycol (spacer 18), or alkane-diol, such as butanediol. Thespacer units are preferably linked by phosphodiester or phosphorothioatebonds. The linker units may appear just once in the molecule or may beincorporated several times, e.g. via phosphodiester, phosphorothioate,methylphosphonate, or amide linkages.

Typical conjugation protocols involve the synthesis of polynucleotidesbearing an aminolinker at one or more positions of the sequence,however, a linker is not required. The amino group is then reacted withthe molecule being conjugated using appropriate coupling or activatingreagents. The conjugation reaction may be performed either with thepolynucleotide still bound to a solid support or following cleavage ofthe polynucleotide in solution phase. Purification of the modifiedpolynucleotide by HPLC typically results in a pure material.

In some embodiments, a linking group can be attached to a nucleomonomerand the transporting peptide can be covalently attached to the linker.In some embodiments, a linker can function as both an attachment sitefor a transporting peptide and can provide stability against nucleases.Examples of suitable linkers include substituted or unsubstituted C₁-C₂₀alkyl chains, C₂-C₂₀ alkenyl chains, C₂-C₂₀ alkynyl chains, peptides,and heteroatoms (e.g., S, O, NH, etc.). Other exemplary linkers includebifunctional crosslinking agents such assulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g., Smithet al. Biochem J 1991.276: 417-2).

In some embodiments, provided oligonucleotides of the invention aresynthesized as molecular conjugates which utilize receptor-mediatedendocytotic mechanisms for delivering genes into cells (see, e.g.,Bunnell et al. 1992. Somatic Cell and Molecular Genetics. 18:559, andthe references cited therein).

Targeting Agents

In some embodiments, provided compositions include one or more targetingagents, associated with the composition and/or with individualoligonucleotides therein. Exemplary such targeting agents are describedabove and further herein. The phrases “targeting agent” and “targetingmoiety” are used herein interchangeably.

Delivery of oligonucleotides and/or compositions thereof can often beimproved by targeting the oligonucleotides to a cellular receptor. Thetargeting moieties can be conjugated to the oligonucleotides or attachedto a carrier group (i.e., poly(L-lysine) or liposomes) linked to theoligonucleotides. This method is well suited to cells that displayspecific receptor-mediated endocytosis.

For instance, oligonucleotide conjugates to 6-phosphomannosylatedproteins are internalized 20-fold more efficiently by cells expressingmannose 6-phosphate specific receptors than free oligonucleotides. Theoligonucleotides may also be coupled to a ligand for a cellular receptorusing a biodegradable linker. In another example, the delivery constructis mannosylated streptavidin which forms a tight complex withbiotinylated oligonucleotides. Mannosylated streptavidin was found toincrease 20-fold the internalization of biotinylated oligonucleotides.(Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).

The field of RNA interference has revolutionized the study of biologicalprocesses. These tools include small inhibitory RNA (siRNA), smallhairpin RNA (shRNA) and ribozymes. As RNA interference technology hasgrown, so has the list of validated RNA target sequences and reagentsfor use. Provided compositions and methods embraced by the presentapplication can be readily applied to each of such target sequences. Fora database of validated siRNA target sequences and links to kits andreagents that may be used for RNA interference experiments, see, forexample, http://www.rnainterference.org/index.html. Exemplary siRNAtarget sequences useful for the present invention are provided in theaccompanying Appendix (A).

Immunomodulatory Oligonucleotides

In some embodiments, provided compositions include one or moreoligonucleotides that are or act as immunomodulatory oligonucleotides.

Oligonucleotides utilized in accordance with the invention can beimmunomodulatory agents, i.e., agents that are capable of modulating orregulating an immune response when administered to a subject. Immuneresponses elicited by such an agent can be an innate and/or an adaptiveimmune response. The immune system is divided into a more innate immunesystem, and acquired adaptive immune system of vertebrates, the latterof which is further divided into humoral cellular components. In someembodiments, the immune response can be mucosal. Immunomodulatory motifsdescribed herein can be used in the context of previously describedclasses of immunomodulatory oligonucleotides including ODN classes suchas A class, B class, C class, E class, T class and P class.

In some embodiments of the invention immunomodulatory oligonucleotidesutilized in accordance with the present invention include one or moreimmunostimulatory motifs. In some embodiments, oligonucleotides utilizedin accordance with the present invention include one or more “CpGdinucleotides.” A CpG dinucleotide can be methylated or unmethylated. Animmunostimulatory oligonucleotide containing at least one unmethylatedCpG dinucleotide is an oligonucleotide molecule which contains anunmethylated cytosine-guanine dinucleotide sequence (i.e., anunmethylated 5′ cytidine followed by 3′ guanosine and linked by aphosphate bond) and which activates the immune system; such animmunostimulatory oligonucleotide is a CpG oligonucleotide. CpGoligonucleotides have been described in a number of issued patents,published patent applications, and other publications, including: U.S.Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and6,339,068, the contents of which are incorporated herein by reference.In some embodiments, utilized oligonucleotides are or act as an agonistfor a Toll-like receptor. In some embodiments, utilized oligonucleotidesare or act as an agonist for Toll-like receptor 9 (TLR9). In someembodiments, utilized oligonucleotides are or act as an antagonist for aToll-like receptor. In some embodiments, utilized oligonucleotides areor act as an antagonist for Toll-like receptor 9 (TLR9). In someembodiments, stereo-defined oligonucleotides embraced by the inventionexhibit greater affinity for respective receptor/target, as compared tostereo-random counterpart. In some embodiments, stereo-definedoligonucleotides embraced by the invention elicit one or more immuneresponses, when administered to subjects that meet certain clinicalcriteria, with less degree of variables amongst the population, ascompared to stereo-random counterpart. In some embodiments,stereo-defined oligonucleotides embraced by the invention cause lessdegree of toxic side effects or fewer side effects, when administered tosubjects that meet certain clinical criteria, as compared tostereo-random counterpart.

In some embodiments, immunostimulatory oligonucleotides utilized inaccordance with the present invention are free of CpG dinucleotidemotifs. These oligonucleotides which are free of CpG dinucleotides arereferred to as non-CpG oligonucleotides, and they have non-CpGimmunostimulatory motifs. In some embodiments, these are T-richimmunostimulatory oligonucleotides, such as oligonucleotides having atleast 80% T.

In some embodiments, immunostimulatory oligonucleotides utilized inaccordance with the present invention are B class immunomodulatoryoligonucleotides.

“B class” ODN are potent at activating B cells but are relatively weakin inducing IFN-α and NK cell activation. The B class CpGoligonucleotides typically are fully stabilized and include anunmethylated CpG dinucleotide within certain preferred base contexts.See, e.g., U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371;6,239,116; and 6,339,068.

Another class is potent for inducing IFN-α and NK cell activation but isrelatively weak at stimulating B cells; this class has been termed the“A class”. In some embodiments, immunostimulatory oligonucleotidesutilized in accordance with the present invention are A classimmunomodulatory oligonucleotides. The A class CpG oligonucleotidestypically have stabilized poly-G sequences at 5′ and 3′ ends and apalindromic phosphodiester CpG dinucleotide-containing sequence of atleast 6 nucleotides. See, for example, published patent applicationPCT/US00/26527 (WO 01/22990).

Yet another class of CpG oligonucleotides activates B cells and NK cellsand induces IFN-α; this class has been termed the C-class. In someembodiments, immunostimulatory oligonucleotides utilized in accordancewith the present invention are C class immunomodulatoryoligonucleotides. The “C class” immunostimulatory oligonucleotidescontain at least two distinct motifs have unique and desirablestimulatory effects on cells of the immune system. Some of these ODNhave both a traditional “stimulatory” CpG sequence and a “GC-rich” or“B-cell neutralizing” motif. These combination motif oligonucleotideshave immune stimulating effects that fall somewhere between thoseeffects associated with traditional “class B” CpG ODN, which are stronginducers of B cell activation and dendritic cell (DC) activation, andthose effects associated with a more recently described class of immunestimulatory oligonucleotides (“class A” CpG ODN) which are stronginducers of IFN-α and natural killer (NK) cell activation but relativelypoor inducers of B-cell and DC activation. Krieg A M et al. (1995)Nature 374:546-9; Ballas Z K et al. (1996) J Immunol 157:1840-5;Yamamoto S et al. (1992) J Immunol 48:4072-6. While preferred class BCpG ODN often have phosphorothioate backbones and preferred class A CpGODN have mixed or chimeric backbones, the C class of combination motifimmune stimulatory oligonucleotides may have either stabilized, e.g.,phosphorothioate, chimeric, or phosphodiester backbones, and in somepreferred embodiments, they have semi-soft backbones. This class hasbeen described in U.S. patent application USI 0/224,523 filed on Aug.19, 2002, the entire contents of which is incorporated herein byreference.

In some embodiments, immunostimulatory oligonucleotides utilized inaccordance with the present invention are E class immunomodulatoryoligonucleotides. The “E class” oligonucleotides have an enhancedability to induce secretion of IFN-alpha. These ODN have a lipophilicsubstituted nucleotide analog 5′ and/or 3′ of a YGZ motif. The compoundof the E class formula may be, for example, any of the followinglipophilic substituted nucleotide analogs: a substituted pyrimidine, asubstituted uracil, a hydrophobic T analog, a substituted toluene, asubstituted imidazole or pyrazole, a substituted triazole,5-chloro-uracil, 5-bromouracil, 5-iodo-uracil, 5-ethyl-uracil,5-propyl-uracil, 5-propinyl-uracil, (E)-5-(2-bromovinyl)-uracil, or2,4-difluoro-toluene. E class oligonucleotides are described at least inprovisional patent application U.S. 60/847,811.

In some embodiments, immunostimulatory oligonucleotides utilized inaccordance with the present invention are T class immunomodulatoryoligonucleotides. The “T class” oligonucleotides induce secretion oflower levels of IFN-alpha when not modified as in the ODNs of theinvention and IFN-related cytokines and chemokines than B class or Cclass oligonucleotides, while retaining the ability to induce levels ofIL-10 similar to B class oligonucleotides. T class oligonucleotides aredescribed at least in U.S. patent application Ser. No. 11/099,683, theentire contents of which are hereby incorporated by reference.

In some embodiments, immunostimulatory oligonucleotides utilized inaccordance with the present invention are P class immunomodulatoryoligonucleotides. The “P class” immunostimulatory oligonucleotides haveseveral domains, including a 5TLR activation domain, 2 duplex formingregions and an optional spacer and 3′ tail. This class ofoligonucleotides has the ability in some instances to induce much higherlevels of IFN-α secretion than the C-Class. The P-Class oligonucleotideshave the ability to spontaneously self-assemble into concatamers eitherin vitro and/or in vivo. Without being bound by any particular theoryfor the method of action of these molecules, one potential hypothesis isthat this property endows the P-Class oligonucleotides with the abilityto more highly crosslink TLR9 inside certain immune cells, inducing adistinct pattern of immune activation compared to the previouslydescribed classes of CpG oligonucleotides. Cross-linking of TLR9receptors may induce activation of stronger IFN-α secretion through thetype I IFNR feedback loop in plasmacytoid dendritic cells. P classoligonucleotides are described at least in U.S. application Ser. No.11/706,561.

In some embodiments, immunostimulatory oligonucleotides utilized inaccordance with the present invention are S class immunomodulatoryoligonucleotides. The immunomodulatory oligonucleotides of the instantinvention may be immunosuppressive oligonucleotides. Theimmunomodulatory motifs described above can be used in the context ofpreviously described classes of immunosuppressive oligonucleotidesincluding ODN classes such as the “S class”. Inhibitory, or S class, ODNare useful whenever it is desirable to inhibit immunostimulation.Inhibitory ODN can be used for preventing and treating septic shock,inflammation, allergy, asthma, graft rejection, graft-versus hostdisease (GvHD), autoimmune diseases, Th1- or Th2-mediated diseases,bacterial infections, parasitic infections, spontaneous abortions, andtumors. The inhibitory ODN can be used generally to inhibit activationof all cells expressing the relevant TLRs, and more specifically toinhibit activation of antigen-presenting cells, B cells, plasmacytoiddendritic cells (pDCs), monocytes, monocyte-derived cells, eosinophils,and neutrophils. S class ODN are further described at least in U.S.application Ser. No. 10/977,560.

According to the invention, immunomodulatory oligonucleotides may have abackbone of stabilized internucleotide linkages in addition to thestabilizing FANA purine nucleotide(s) or have a chimeric backbone ofstabilized and phosphodiester nucleotide linkages. A “stabilizedinternucleotide linkage” shall mean an internucleotide linkage that isrelatively resistant to in vivo degradation (e.g., via an exo- orendo-nuclease), compared to a phosphodiester internucleotide linkage. Insome embodiments, stabilized internucleotide linkages include, withoutlimitation, phosphorothioate, phosphorodithioate, methylphosphonate,methylphosphorothioate, phosphonoacetate, Rp-phosphorothioate,Sp-phosphorothioate, boranophosphate, or 3′-thioformacetal, orcombinations thereof. Other stabilized oligonucleotides include:nonionic DNA analogs, such as alkyl- and aryl-phosphates (in which thecharged phosphonate oxygen is replaced by an alkyl or aryl group),phosphodiester and alkylphosphotriesters, in which the charged oxygenmoiety is alkylated. Oligonucleotides which contain diol, such astetraethyleneglycol or hexaethyleneglycol, at either or both terminihave also been shown to be substantially resistant to nucleasedegradation.

As described in more detained herein, according to the invention, theseand other modifications can be selectively introduced at a predeterminedposition(s)/pattern(s) within an oligonucleotide so as to obtain astereospecific oligonucleotide molecule. Furthermore, according to theinvention, a composition comprising a plurality of such oligonucleotidesat an extremely high degree of purity (e.g., at least 96%, at least 97%,at least 98%, at least 99%, essentially 100%) can be obtained. Thus,provided oligonucleotide compositions comprising oligonucleotides of oneor more predetermined types are suitable for in vivo administration. Itis contemplated that, due to high degree of structural purity ofoligonucleotides in a composition (such that a composition comprises asingle specified type of oligonucleotides), provided compositions canelicit improved biological activities, increased efficacy, reducedvariables in response, and/or reduced unwanted side effects, whenadministered to subjects.

Accordingly, utilized oligonucleotides of the invention are particularlyuseful as an therapeutic agent when formulated into a pharmaceuticalcomposition. In some embodiments, such therapeutic agent is animmunomodulatory agent. In some embodiments, provided immunomodulatoryagents are immunostimulatory agents. In some embodiments, providedimmunomodulatory agents are immunoinhibitory (or immunosuppressive)agents. In some embodiments, provided immunomodulatory agents act as anadjuvant. In some embodiments, provided immunomodulatory agents canshift a Th2 type immune response to a Th1 type immune response byinducing Th1 type cytokines that can suppress inflammatory Th2 response.In some embodiments, provided oligonucleotide compositions are useful asan agent that regulates gene expression. For example, in someembodiments, provided oligonucleotide compositions are useful as anagent capable of silencing a gene of interest, e.g., genes associatedwith disease or disorder. In some embodiments, provided oligonucleotidecompositions are useful as an agent to regulate RNA splicing.

For example, some embodiments of such oligonucleotides include thosecomprising at least one CpG dinucleotide motif. In some embodiments,such oligonucleotides comprise at least one unmethylated CpGdinucleotide motif. In some embodiments, CpG-containing oligonucleotidesuseful for the present invention are categorized as Class A (type D)oligonucleotides. In some embodiments, CpG-containing oligonucleotidesuseful for the present invention are categorized as Class B (type K)oligonucleotides. In some embodiments, CpG-containing oligonucleotidesuseful for the present invention are categorized as Class Coligonucleotides. In some embodiments, CpG-containing oligonucleotidesuseful for the present invention are categorized as Class Poligonucleotides. In some embodiments, CpG-containing oligonucleotidesuseful for the present invention contain at least one palindromicsequence. In some embodiments, CpG-containing oligonucleotides usefulfor the present invention can form a “dumbbell-like” structure. In someembodiments, CpG-containing oligonucleotides useful for the presentinvention can form a “Y” shaped structure, or multimers thereof. In someembodiments, CpG-containing oligonucleotides useful for the presentinvention can form a tetrahedral structure. For reviews, see, forexample: Bode et al. (2011) Expert Rev. Vaccines 10(4): 499-511;Hanagata (2012) Int. J. Nanomedicine 7:2181-95.

In some embodiments, oligonucleotides utilized in accordance with thepresent invention elicit immunomodulatory effects on cells expressingToll-like receptor(s). In some embodiments, target cells that respond tosuch oligonucleotides include, but are not limited to,antigen-presenting cells (APCs), antigen-specific T and B cells,cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and dendriticcells (DCs). In some embodiments, immunomodulatory effects are thedirect effects elicited by cells expressing a receptor or receptors thatrecognize such an oligonucleotide. In some embodiments, sucholigonucleotides elicit immunomodulatory effects on cells expressingToll-like receptor 9 (TLR9) as ligands. For example, some embodiments ofthe invention include CpG oligonucleotides that act as agonists of oneor more Toll-like receptors. Some embodiments of the invention includeCpG oligonucleotides that act as antagonists of one or more Toll-likereceptors. In some embodiments, immunomodulatory effects are indirecteffects that occur downstream, involving cells that do not necessarilyrespond directly to such oligonucleotides or express such receptors.

In some embodiments, CpG oligonucleotides useful for the presentinvention are characterized in that they directly activate human B cellsand plasmacytoid dendritic cells via TLR-9. In some embodiments, CpGoligodeoxynucleotides useful for the present invention are characterizedin that indirectly support the maturation and proliferation of naturalkiller cells, T cells and monocytes/macrophages.

In some embodiments, CpG oligonucleotides useful for the presentinvention trigger an immune response, which is characterized by theproduction of Th1-type and proinflammatory cytokines, chemokines andpolyreactive IgM.

In some embodiments, CpG oligonucleotides useful for the presentinvention are characterized in that the immunogenicity of conventionalprotein antigens and peptide-based vaccines is enhanced by sucholigonucleotides. Without wising to be bound by a particular theory, itis believed that such adjuvant effect is mediated through improvedfunction of professional antigen-presenting cells and the resultantgeneration of humoral and cellular vaccine-specific immune responses.

In some embodiments, CpG oligonucleotides useful for the presentinvention are characterized in that CpG oligonucleotides increase themagnitude and accelerate the development of vaccineinduced responses.They also improve the induction of memory, thereby extending theduration of humoral and cellular immunity.

In some embodiments, CpG oligonucleotides useful for the presentinvention are characterized in that they boost immunity in groups ofsubjects (e.g., populations of patients) with reduced immune function,such as the elderly and those with suppressed immune system. They areeffective when administered either systemically or mucosally.Preclinical and clinical trials using CpG oligonucleotides of mixedchirality (e.g., not chirally pure) demonstrate CpG oligonucleotides canboost the immunogenicity of vaccines targeting infectious diseases andcancer. Furthermore, clinical trials indicate that CpG oligonucleotidesare reasonably safe when administered s vaccine adjuvants.

Immunomodulatory oligonucleotides prepared in accordance with thepresent disclosure are useful for a number of therapeutic applications.Therefore, utilized immunomodulatory oligonucleotides may be formulatedinto suitable pharmaceutical compositions. Such pharmaceuticalcompositions can be administered to a subject in an amount effective totreat a disease, disorder or condition, as described in further detailherein by suitable routes of administration. According to the invention,an effective amount of an immunostimulatory oligonucleotides may beadministered to a subject who is likely to benefit from boosting theimmune system (e.g., enhanced immune response). In some embodiments,clinical benefits are conferred directly by an immunomodulatoryoligonucleotide acting upon its target immune cells, e.g., via bindingof the ligand to its Toll-like receptor proteins. In some embodiments,clinical benefits are achieved at least in part indirectly by overallboosting of the immune system of the subject.

As used herein, the terms “effective amount” and “effective dose” referto any amount or dose of a compound or composition that is sufficient tofulfill its intended purpose(s), i.e., a desired biological or medicinalresponse in a tissue or subject at an acceptable benefit/risk ratio. Therelevant intended purpose may be objective (i.e., measurable by sometest or marker) or subjective (i.e., subject gives an indication of orfeels an effect). In some embodiments, a therapeutically effectiveamount is an amount that, when administered to a population of subjectsthat meet certain clinical criteria for a disease or disorder (forexample, as determined by symptoms manifested, diseaseprogression/stage, genetic profile, etc.), a statistically significanttherapeutic response is obtained among the population. A therapeuticallyeffective amount is commonly administered in a dosing regimen that maycomprise multiple unit doses. For any particular pharmaceutical agent, atherapeutically effective amount (and/or an appropriate unit dose withinan effective dosing regimen) may vary, for example, depending on routeof administration, on combination with other pharmaceutical agents. Insome embodiments, the specific therapeutically effective amount (and/orunit dose) for any particular patient may depend upon a variety offactors including the disorder being treated and the severity of thedisorder; the activity of the specific pharmaceutical agent employed;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration, route ofadministration, and/or rate of excretion or metabolism of the specificpharmaceutical agent employed; the duration of the treatment; and likefactors as is well known in the medical arts. Those of ordinary skill inthe art will appreciate that in some embodiments of the invention, aunit dosage may be considered to contain an effective amount if itcontains an amount appropriate for administration in the context of adosage regimen correlated with a positive outcome. In some embodiments,CpG oligodeoxynucleotides prepared according to the present inventiontherefore may exert improved efficacy and immunomodulatory effects dueto their chiral purity. In some embodiments, an effective amount ofprovided immunomodulatory oligonucleotides is less than that ofless-pure counterpart.

In some embodiments, an immunomodulatory oligonucleotide composition isadministered to a healthy subject. In some embodiments, animmunostimulatory oligonucleotide composition is administered to asubject as part of a vaccine, in which the immunostimulatoryoligonucleotide acts as an adjuvant. In some embodiments, animmunostimulatory oligonucleotide composition is administered to asubject with a suppressed (e.g., compromised) immune system. In someembodiments, a subject, whose immune system is suppressed and does notsufficiently respond to a conventional vaccine, responds to a vaccinecomprising an immunostimulatory oligonucleotide embraced by the presentinvention. In some embodiments, a subject who may benefit from such avaccine has a suppressed immune system associated with an infection,such as viral infection, e.g., HIV, HBV, HCV, etc.

The invention encompasses the use of immunomodulatory oligonucleotidescontemplated herein for treatment of a subject having a condition thatmay be treated by stimulation or suppression of the immune response.Thus, provided immunomodulatory oligonucleotide compositions are usefulfor the treatment of diseases or conditions, including, withoutlimitation, infection, cancer, allergy, asthma, an inflammatorycondition, an autoimmune disease, and any combination thereof.

In some embodiments, provided immunomodulatory oligonucleotidecompositions are useful in some aspects of the invention for thetreatment of a subject at risk of developing a clinical condition.Non-limiting clinical conditions include allergy, asthma, an infectionwith an infectious organism, cancer, inflammation, and autoimmunedisease. A subject at risk as used herein is a subject who has any riskof exposure to an infection causing pathogen or a cancer or an allergenor a risk of developing cancer. For instance, a subject at risk may be asubject who is planning to travel to an area where a particular type ofinfectious agent is found, or it may be a subject who through lifestyleor medical procedures is exposed to bodily fluids which may containinfectious organisms or directly to the organism, or even any subjectliving in an area where an infectious organism or an allergen has beenidentified. Subjects at risk of developing infection also includegeneral populations to which a medical agency recommends vaccinationwith a particular infectious organism antigen. If the antigen is anallergen and the subject develops allergic responses to that particularantigen and the subject may be exposed to the antigen, i.e., duringpollen season, then that subject is at risk of exposure to the antigen.A subject at risk of developing allergy or asthma includes thosesubjects that have been identified as having an allergy or asthma butthat don't have the active disease during the immunomodulatoryoligonucleotide treatment as well as subjects that are considered to beat risk of developing these diseases because of genetic or environmentalfactors. A subject at risk of developing a cancer is one who has a highprobability of developing cancer. These subjects include, for instance,subjects having a genetic abnormality, the presence of which has beendemonstrated to have a correlative relation to a higher likelihood ofdeveloping a cancer and subjects exposed to cancer causing agents suchas tobacco, asbestos, or other chemical toxins, or a subject who haspreviously been treated for cancer and is in apparent remission. When asubject at risk of developing a cancer is treated with an antigenspecific for the type of cancer to which the subject is at risk ofdeveloping and a CpG immunostimulatory oligonucleotide, the subject maybe able to kill the cancer cells as they develop. If a tumor begins toform in the subject, the subject will develop a specific immune responseagainst the tumor antigen.

In some embodiments, provided immunomodulatory oligonucleotidecompositions are useful for treating a subject having an immune diseaseor disorder, including asthma, allergy, and related conditions.

A subject having an allergy is a subject that has or is at risk ofdeveloping an allergic reaction in response to an allergen. An allergyrefers to acquired hypersensitivity to a substance (allergen). Allergicconditions include but are not limited to eczema, allergic rhinitis orcoryza, hay fever, conjunctivitis, bronchial asthma, urticaria (hives)and food allergies, and other atopic conditions.

Allergies are generally caused by IgE antibody generation againstharmless allergens. The cytokines that are induced by systemic ormucosal administration of immunomodulatory oligonucleotides arepredominantly of a class called Th1 (examples are IL-12, IP-10, IFN-αand IFN-γ) and these induce both humoral and cellular immune responses.The other major type of immune response, which is associated with theproduction of IL-4 and IL-5 cytokines, is termed a Th2 immune response.In general, it appears that allergic diseases are mediated by Th2 typeimmune responses. Based on the ability of the immunomodulatoryoligonucleotide to shift the immune response in a subject from apredominant Th2 (which is associated with production of IgE antibodiesand allergy) to a balanced Th2/Th1 response (which is protective againstallergic reactions), an effective dose for inducing an immune responseof a immunomodulatory oligonucleotide can be administered to a subjectto treat or prevent asthma and allergy.

Thus, provided immunomodulatory oligonucleotide compositions havesignificant therapeutic utility in the treatment of allergic andnon-allergic conditions such as asthma. Th2 cytokines, especially IL-4and IL-5 are elevated in the airways of asthmatic subjects. Thesecytokines promote important aspects of the asthmatic inflammatoryresponse, including IgE isotope switching, eosinophil chemotaxis andactivation and mast cell growth. Th1 cytokines, especially IFN-γ andIL-12, can suppress the formation of Th2 clones and production of Th2cytokines. Asthma refers to a disorder of the respiratory systemcharacterized by inflammation, narrowing of the airways and increasedreactivity of the airways to inhaled agents. Asthma is frequently,although not exclusively associated with atopic or allergic symptoms.

Provided immunomodulatory oligonucleotide compositions may also beadministered in conjunction with an anti-allergy therapy. Conventionalmethods for treating or preventing allergy have involved the use ofallergy medicaments or desensitization therapies. Some evolvingtherapies for treating or preventing allergy include the use ofneutralizing anti-lgE antibodies. Anti-histamines and other drugs whichblock the effects of chemical mediators of the allergic reaction help toregulate the severity of the allergic symptoms but do not prevent theallergic reaction and have no effect on subsequent allergic responses.Desensitization therapies are performed by giving small doses of anallergen, usually by injection under the skin, in order to induce anIgG-type response against the allergen. The presence of IgG antibodyhelps to neutralize the production of mediators resulting from theinduction of IgE antibodies, it is believed. Initially, the subject istreated with a very low dose of the allergen to avoid inducing a severereaction and the dose is slowly increased. This type of therapy isdangerous because the subject is actually administered the compoundswhich cause the allergic response and severe allergic reactions canresult.

Anti-allergy medicaments include, but are not limited to,anti-histamines, corticosteroids, and prostaglandin inducers.Anti-histamines are compounds which counteract histamine released bymast cells or basophils. These compounds are well known in the art andcommonly used for the treatment of allergy. Anti-histamines include, butare not limited to, acrivastine, astemizole, azatadine, azelastine,betatastine, brompheniramine, buclizine, cetirizine, cetirizineanalogues, chlorpheniramine, clemastine, CS 560, cyproheptadine,desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine,HSR 609, hydroxyzine, levocabastine, loratidine, methscopolamine,mizolastine, norastemizole, phenindamine, promethazine, pyrilamine,terfenadine, and tranilast. Corticosteroids include, but are not limitedto, methylprednisolone, prednisolone, prednisone, beclomethasone,budesonide, dexamethasone, flunisolide, fluticasone propionate, andtriamcinolone. Although dexamethasone is a corticosteroid havingantiinflammatory action, it is not regularly used for the treatment ofallergy or asthma in an inhaled form because it is highly absorbed andit has long-term suppressive side effects at an effective dose.Dexamethasone, however, can be used according to the invention fortreating allergy or asthma because when administered in combination witha composition of the invention it can be administered at a low dose toreduce the side effects. Some of the side effects associated withcorticosteroid use include cough, dysphonia, oral thrush (candidiasis),and in higher doses, systemic effects, such as adrenal suppression,glucose intolerance, osteoporosis, aseptic necrosis of bone, cataractformation, growth suppression, hypertension, muscle weakness, skinthinning, and easy bruising. Barnes & Peterson (1993) Am Rev Respir Dis148:S1-S26; and Kamada A K et al. (1996) Am J Respir Crit Care Med153:1739-48.

Provided oligonucleotide compositions and methods in accordance with theinvention can be used alone or in conjunction with other agents andmethods useful for the treatment of asthma. In one aspect the inventionprovides a method of treating a subject having asthma. The methodaccording to this aspect of the invention includes the step ofadministering to a subject having asthma an effective amount of acomposition of the invention to treat the subject.

In some embodiments, the invention provides a method of treating asubject having asthma. The method according to this aspect of theinvention includes the step of administering to a subject having asthmaan effective amount of the composition of the invention and ananti-asthma therapy to treat the subject.

“Asthma” as used herein refers to a disorder of the respiratory systemcharacterized by inflammation and narrowing of the airways, andincreased reactivity of the airways to inhaled agents. Asthma isfrequently, although not exclusively, associated with an atopic orallergic condition. Symptoms of asthma include recurrent episodes ofwheezing, breathlessness, chest tightness, and coughing, resulting fromairflow obstruction. Airway inflammation associated with asthma can bedetected through observation of a number of physiological changes, suchas, denudation of airway epithelium, collagen deposition beneathbasement membrane, edema, mast cell activation, inflammatory cellinfiltration, including neutrophils, eosinophils, and lymphocytes. As aresult of the airway inflammation, asthma patients often experienceairway hyper-responsiveness, airflow limitation, respiratory symptoms,and disease chronicity. Airflow limitations include acutebronchoconstriction, airway edema, mucous plug formation, and airwayremodeling, features which often lead to bronchial obstruction. In somecases of asthma, sub-basement membrane fibrosis may occur, leading topersistent abnormalities in lung function.

Research over the past several years has revealed that asthma likelyresults from complex interactions among inflammatory cells, mediators,and other cells and tissues resident in the airways. Mast cells,eosinophils, epithelial cells, macrophage, and activated T cells allplay an important role in the inflammatory process associated withasthma. Djukanovic R et al. (1990) Am Rev Respir Dis 142:434-457. It isbelieved that these cells can influence airway function throughsecretion of preformed and newly synthesized mediators which can actdirectly or indirectly on the local tissue. It has also been recognizedthat subpopulations of T lymphocytes (Th2) play an important role inregulating allergic inflammation in the airway by releasing selectivecytokines and establishing disease chronicity. Robinson D S et al.(1992) N Engl J Med 326:298-304.

Asthma is a complex disorder which arises at different stages indevelopment and can be classified based on the degree of symptoms asacute, subacute, or chronic. An acute inflammatory response isassociated with an early recruitment of cells into the airway. Thesubacute inflammatory response involves the recruitment of cells as wellas the activation of resident cells causing a more persistent pattern ofinflammation. Chronic inflammatory response is characterized by apersistent level of cell damage and an ongoing repair process, which mayresult in permanent abnormalities in the airway.

A “subject having asthma” is a subject that has a disorder of therespiratory system characterized by inflammation and narrowing of theairways and increased reactivity of the airways to inhaled agents.Factors associated with initiation of asthma include, but are notlimited to, allergens, cold temperature, exercise, viral infections, andSO₂.

As mentioned above, asthma may be associated with a Th2-type of immuneresponse, which is characterized at least in part by Th2 cytokines IL-4and IL-5, as well as antibody isotype switching to IgE. Th1 and Th2immune responses are mutually counter-regulatory, so that skewing of theimmune response toward a Th1-type of immune response can prevent orameliorate a Th2-type of immune response, including allergy. Providedimmunomodulatory oligonucleotide compositions of the invention aretherefore useful by themselves to treat a subject having asthma becausethe analogs can skew the immune response toward a Th1-type of immuneresponse.

Provided immunomodulatory oligonucleotide compositions of the inventionmay also be administered in conjunction with an asthma therapy.Conventional methods for treating or preventing asthma have involved theuse of anti-allergy therapies (described above) and a number of otheragents, including inhaled agents.

Medications for the treatment of asthma are generally separated into twocategories, quick-relief medications and long-term control medications.Asthma patients take the long-term control medications on a daily basisto achieve and maintain control of persistent asthma. Long-term controlmedications include anti-inflammatory agents such as corticosteroids,chromolyn sodium and nedocromil; long-acting bronchodilators, such aslong-acting β₂-agonists and methylxanthines; and leukotriene modifiers.The quick-relief medications include short-acting β₂ agonists,anticholinergics, and systemic corticosteroids. There are many sideeffects associated with each of these drugs and none of the drugs aloneor in combination is capable of preventing or completely treatingasthma.

Anti-asthma medicaments include, but are not limited to, PDE-4inhibitors, bronchodilator/beta-2 agonists, K+ channel openers, VLA-4antagonists, neurokin antagonists, thromboxane A2 (TXA2) synthesisinhibitors, xanthines, arachidonic acid antagonists, 5 lipoxygenaseinhibitors, TXA2 receptor antagonists, TXA2 antagonists, inhibitor of5-lipox activation proteins, and protease inhibitors.

Bronchodilator/β₂ agonists are a class of compounds which causebronchodilation or smooth muscle relaxation. Bronchodilator/β₂ agonistsinclude, but are not limited to, salmeterol, salbutamol, albuterol,terbutaline, D2522/formoterol, fenoterol, bitolterol, pirbuerolmethylxanthines and orciprenaline. Long-acting β₂ agonists andbronchodilators are compounds which are used for long-term prevention ofsymptoms in addition to the anti-inflammatory therapies. Long-acting β₂agonists include, but are not limited to, salmeterol and albuterol.These compounds are usually used in combination with corticosteroids andgenerally are not used without any inflammatory therapy. They have beenassociated with side effects such as tachycardia, skeletal muscletremor, hypokalemia, and prolongation of QTc interval in overdose.

Methylxanthines, including for instance theophylline, have been used forlong-term control and prevention of symptoms. These compounds causebronchodilation resulting from phosphodiesterase inhibition and likelyadenosine antagonism. Dose-related acute toxicities are a particularproblem with these types of compounds. As a result, routine serumconcentration must be monitored in order to account for the toxicity andnarrow therapeutic range arising from individual differences inmetabolic clearance. Side effects include tachycardia, tachyarrhythmias,nausea and vomiting, central nervous system stimulation, headache,seizures, hematemesis, hyperglycemia and hypokalemia. Short-acting β₂agonists include, but are not limited to, albuterol, bitolterol,pirbuterol, and terbutaline. Some of the adverse effects associated withthe administration of short-acting β₂ agonists include tachycardia,skeletal muscle tremor, hypokalemia, increased lactic acid, headache,and hyperglycemia.

Chromolyn sodium and nedocromil are used as long-term controlmedications for preventing primarily asthma symptoms arising fromexercise or allergic symptoms arising from allergens. These compoundsare believed to block early and late reactions to allergens byinterfering with chloride channel function. They also stabilize mastcell membranes and inhibit activation and release of mediators frominosineophils and epithelial cells. A four to six week period ofadministration is generally required to achieve a maximum benefit.

Anticholinergics are generally used for the relief of acutebronchospasm. These compounds are believed to function by competitiveinhibition of muscarinic cholinergic receptors. Anticholinergicsinclude, but are not limited to, ipratropium bromide. These compoundsreverse only cholinerigically-mediated bronchospasm and do not modifyany reaction to antigen. Side effects include drying of the mouth andrespiratory secretions, increased wheezing in some individuals, andblurred vision if sprayed in the eyes.

In some embodiments, provided immunomodulatory oligonucleotidecompositions may also be useful for treating airway remodeling. Airwayremodeling results from smooth muscle cell proliferation and/orsubmucosal thickening in the airways, and ultimately causes narrowing ofthe airways leading to restricted airflow. The immunomodulatoryoligonucleotides of the invention may prevent further remodeling andpossibly even reduce tissue build-up resulting from the remodelingprocess.

In some embodiments, provided immunomodulatory oligonucleotidecompositions are useful for treating a subject having an inflammatorydisorder. As used herein, the term “inflammatory disorder” refers to acondition associated with an antigen-nonspecific reaction of the innateimmune system that involves accumulation and activation of leukocytesand plasma proteins at a site of infection, toxin exposure, or cellinjury. Cytokines that are characteristic of inflammation include tumornecrosis factor (TNF-α), interleukin 1 (IL-1), IL-6, IL-12, interferonalpha (IFN-α), interferon beta (IFN-β), and chemokines. Thus, certaintypes of asthma, allergy, and autoimmune disorders may havecharacteristics of an inflammatory disorder. Inflammatory disorders alsoinclude, for example cardiovascular disease, chronic obstructivepulmonary disease (COPD), bronchiectasis, chronic cholecystitis,tuberculosis, Hashimoto's thyroiditis, sepsis, sarcoidosis, silicosisand other pneumoconiosis, and an implanted foreign body in a wound, butare not so limited. As used herein, the term “sepsis” refers to awell-recognized clinical syndrome associated with a host's systemicinflammatory response to microbial invasion. The term “sepsis” as usedherein refers to a condition that is typically signaled by fever orhypothermia, tachycardia, and tachypnea, and in severe instances canprogress to hypotension, organ dysfunction, and even death.

In some embodiments, provided immunomodulatory oligonucleotidecompositions are useful for treating a subject having an infection. Insome embodiments, provided immunomodulatory oligonucleotide compositionsare useful for treating a subject who is susceptible to an infection,including those who may have exposure to a pathogen or pathogens. Theimmunomodulatory oligonucleotides utilized in accordance with theinvention can in some aspects also be used to treat or preventinfections by viruses, bacteria, fungi, or parasites. A subject havingan infection is a subject that has been exposed to an infectiouspathogen and has acute or chronic detectable levels of the pathogen inthe body. The immunomodulatory oligonucleotides can be used with orwithout an antigen to mount an antigen specific systemic or mucosalimmune response that is capable of reducing the level of or eradicatingthe infectious pathogen. An infectious disease, as used herein, is adisease arising from the presence of a foreign microorganism in thebody. It is particularly important to develop effective vaccinestrategies and treatments to protect the body's mucosal surfaces, whichare the primary site of pathogenic entry.

Viruses are small infectious agents which generally contain a nucleicacid core and a protein coat, but are not independently livingorganisms. Viruses can also take the form of infectious nucleic acidslacking a protein. A virus cannot survive in the absence of a livingcell within which it can replicate. Viruses enter specific living cellseither by endocytosis or direct injection of DNA (phage) and multiply,causing disease. The multiplied virus can then be released and infectadditional cells. Some viruses are DNA-containing viruses and others areRNA-containing viruses. DNA viruses include Pox, Herpes, Adeno, Papova,Parvo, and Hepadna. RNA viruses include Picorna, Calici, Astro.Toga,Flavi, Corona, Paramyxo, Orthomyxo, Bunya, Arena, Rhabdo, FiIo, Borna,Reo, and Retro. In some aspects, the invention also intends to treatdiseases in which prions are implicated in disease progression such asfor example bovine spongiform encephalopathy (i.e., mad cow disease,BSE) or scrapie infection in animals, or Creutzfeldt-Jakob disease inhumans.

Viruses include, but are not limited to, enteroviruses (including, butnot limited to, viruses that the family picornaviridae, such as poliovirus, Coxsackie virus, echo virus), rotaviruses, adenovirus, andhepatitis virus, such as hepatitis A, B, C D and E. Specific examples ofviruses that have been found in humans include but are not limited to:Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (alsoreferred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and otherisolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitisA virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g., strains that cause gastroenteritis);Togaviridae (e.g., equine encephalitis viruses, rubella viruses);Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (e.g., coronavi ruses); Rhabdoviridae (e.g.,vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebolaviruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.,influenza viruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovavihdae (papillomaviruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV));Poxviridae (variola viruses, vaccinia viruses, pox viruses);Iridoviridae (e.g., African swine fever virus); and other viruses acutelaryngotracheobronchitis virus, Alphavirus, Kaposi's sarcoma-associatedherpesvirus, Newcastle disease virus, Nipah virus, Norwalk virus,Papillomavirus, parainfluenza virus, avian influenza, SARs virus, WestNile virus. Viruses that infect plants include, for example, viruses ofthe genera and/or families of Alfamoviruses: Bromoviridae,Alphacryptoviruses: Partitiviridae, Badnaviruses, Betacryptoviruses:Partitiviridae, Bigeminiviruses: Geminiviridae, Bromoviruses:Bromoviridae, Bymoviruses: Potyviridae, Capilloviruses, Carlaviruses,Carmoviruses: Tombusviridae, Caulimoviruses, Closteroviruses,Comoviruses: Comoviridae, Cucumoviruses: Bromoviridae,Cytorhabdoviruses: Rhabdoviridae, Dianthoviruses, Enamoviruses,Fabaviruses: Comoviridae, Fijiviruses: Reoviridae, Furoviruses,Hordeiviruses, Hybrigeminiviruses: Geminiviridae, Idaeoviruses,Ilarviruses: Bromoviridae, Ipomoviruses: Potyviridae, Luteoviruses,Machlomoviruses, Macluraviruses, Marafiviruses, Monogeminiviruses:Geminiviridae, Nanaviruses, Necroviruses, Nepoviruses: Comoviridae,Nucleorhabdoviruses: Rhabdoviridae, Oryzaviruses: Reoviridae,Ourmiaviruses, Phytoreoviruses: Reoviridae, Potexviruses, Potyviruses:Potyviridae, Rymoviruses: Potyviridae, Satellite RNAs, Satelliviruses,Sequiviruses: Sequiviridae, Sobemoviruses, Tenuiviruses, Tobamoviruses,Tobraviruses, Tombusviruses: Tombusviridae, Tospoviruses: Bunyaviridae,Trichoviruses, Tymoviruses, Umbraviruses, Unassigned potyviruses:Potyviridae, Unassigned rhabdoviruses: Rhabdoviridae, Varicosaviruses,Waikaviruses: Sequiviridae, Ungrouped viruses, Bromoviridae Potyviridaeand Tymoviridae; particular relevant plant viruses include, for example,Tobacco mosaic virus (TMV), Tomato spotted wilt virus, Tomato yellowleaf curl virus, Cucumber mosaic virus, Potato virus Y, Cauliflowermosaic virus, African cassava mosaic virus, Plum pox virus, Brome mosaicvirus, Potato virus X, Citrus tristeza virus, Barley yellow dwarf virus,Potato leafroll virus and Tomato bushy stunt virus.

Viral hepatitis is an inflammation of the liver which may produceswelling, tenderness, and sometimes permanent damage to the liver. Ifthe inflammation of the liver continues at least six months or longer,it is referred to as chronic hepatitis. There are at least fivedifferent viruses known to cause viral hepatitis, include hepatitis A₁B,C D and E. Hepatitis A is generally communicated through food ordrinking water contaminated with human feces. Hepatitis B generally isspread through bodily fluids such as blood. For instance, it may bespread from mother to child at birth, through sexual contact,contaminated blood transfusions and needles. Hepatitis C is quite commonand like Hepatitis B is often spread through blood transfusions andcontaminated needles. Hepatitis D is found most often in IV drug userswho are carriers of the hepatitis B virus with which it co-associates.Hepatitis E is similar to viral hepatitis A and is generally associatedwith poor sanitation.

Both gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to, Pasteυrella species, Staphylococci species, andStreptococcus species. Gram negative bacteria include, but are notlimited to, Escherichia coli, Pseudomonas species, and Salmonellaspecies. Specific examples of infectious bacteria include but are notlimited to, Helicobacter pyloris, Borelia burgdorferi, Legionellapneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M.intracellular, M. kansaii, M. gordonae), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Haemophilus influenzae, Bacillus antracis, Corynebacteriumdiphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae,Clostridium pen ‘ringers, Clostridium tetani, Enterobacter aerogenes,Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp.,Fusobacterium nucleatum,

Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue,Leptospira, Rickettsia, and Actinomyces israelii.

Examples of fungi include Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, Candida albicans.

Other infectious organisms (i.e., protists) include Plasmodium spp. suchas Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, andPlasmodium vivax and Toxoplasma gondii. Blood-borne and/or tissuesparasites include Plasmodium spp., Babesia microti, Babesia divergens,Leishmania tropica, Leishmania spp., Leishmania braziliensis, Leishmaniadonovani, Trypanosoma gambiense and Trypanosoma rhodesiense (Africansleeping sickness), Trypanosoma cruzi (Chagas' disease), and Toxoplasmagondii.

Other medically relevant microorganisms have been described extensivelyin the literature, e.g., see C. G. A Thomas, Medical Microbiology,Bailliere Tindall, Great Britain 1983, the entire contents of which ishereby incorporated by reference. Agriculturally relevant microorganisms(e.g., those that infect crops and/or livestock) are also known, forexample but not limited to those described in, e.g., George N. Agrios,Plant Pathology, Elsevier Academic Press, 5^(th) Edition, 2005; JohnLucas, Plant Pathology and Plant Pathogens, Wiley-Blackwell; 3^(rd)edition, 1998; Dwight C. Hirsh, N. James MacLachlan, Richard L. Walker,Veterinary Microbiology, Wiley-Blackwell; 2 edition, 2004; and P. J.Quinn, et al, Veterinary Microbiology and Microbial Disease,Wiley-Blackwell; 2 edition, 2011.

Provided oligonucleotide compositions of the invention may beadministered to a subject (including, for example, a human subject or,in some embodiments an animal [e.g., livestock or pet animal] or plant[e.g., crop] subject) with an anti-microbial agent. An anti-microbialagent, as used herein, refers to a naturally-occurring or syntheticcompound which is capable of killing or inhibiting infectiousmicroorganisms. The type of anti-microbial agent useful according to theinvention will depend upon the type of microorganism with which thesubject is infected or at risk of becoming infected. Anti-microbialagents include but are not limited to anti-bacterial agents, anti-viralagents, anti-fungal agents and anti-parasitic agents. Phrases such as“anti-infective agent”, “anti-bacterial agent”, “anti-viral agent”,“anti-fungal agent”, “antiparasitic agent” and “parasiticide” havewell-established meanings to those of ordinary skill in the art and aredefined in standard medical texts. Briefly, anti-bacterial agents killor inhibit bacteria, and include antibiotics as well as other syntheticor natural compounds having similar functions. Antibiotics are lowmolecular weight molecules which are produced as secondary metabolitesby cells, such as microorganisms. In general, antibiotics interfere withone or more bacterial functions or structures which are specific for themicroorganism and which are not present in host cells. Anti-viral agentscan be isolated from natural sources or synthesized and are useful forkilling or inhibiting viruses. Anti-fungal agents are used to treatsuperficial fungal infections as well as opportunistic and primarysystemic fungal infections. Anti-parasite agents kill or inhibitparasites.

Examples of anti-parasitic agents, also referred to as parasiticidesuseful for human administration include but are not limited toalbendazole, amphotericin B, benznidazole, bithionol, chloroquine HCI,chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine,diloxanide furoate, eflornithine, furazolidaone, glucocorticoids,halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumineantimoniate, melarsoprol, metrifonate, metronidazole, niclosamide,nifurtimox, oxamniquine, paromomycin, pentamidine isethionate,piperazine, praziquantel, primaquine phosphate, proguanil, pyrantelpamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine,quinacrine HCI, quinine sulfate, quinidine gluconate, spiramycin,stibogluconate sodium (sodium antimony gluconate), suramin,tetracycline, doxycycline, thiabendazole, tinidazole,trimethroprim-sulfamethoxazole, and tryparsamide some of which are usedalone or in combination with others.

Antibacterial agents kill or inhibit the growth or function of bacteria.A large class of antibacterial agents is antibiotics. Antibiotics, whichare effective for killing or inhibiting a wide range of bacteria, arereferred to as broad spectrum antibiotics. Other types of antibioticsare predominantly effective against the bacteria of the classgram-positive or gram-negative. These types of antibiotics are referredto as narrow spectrum antibiotics. Other antibiotics which are effectiveagainst a single organism or disease and not against other types ofbacteria, are referred to as limited spectrum antibiotics. Antibacterialagents are sometimes classified based on their primary mode of action.In general, antibacterial agents are cell wall synthesis inhibitors,cell membrane inhibitors, protein synthesis inhibitors, nucleic acidsynthesis or functional inhibitors, and competitive inhibitors.

Antiviral agents are compounds which prevent infection of cells byviruses or replication of the virus within the cell. There are manyfewer antiviral drugs than antibacterial drugs because the process ofviral replication is so closely related to DNA replication within thehost cell, that non-specific antiviral agents would often be toxic tothe host. There are several stages within the process of viral infectionwhich can be blocked or inhibited by antiviral agents. These stagesinclude, attachment of the virus to the host cell (immunoglobulin orbinding peptides), uncoating of the virus (e.g. amantadine), synthesisor translation of viral mRNA (e.g. interferon), replication of viral RNAor DNA (e.g. nucleotide analogues), maturation of new virus proteins(e.g. protease inhibitors), and budding and release of the virus.

Nucleotide analogues are synthetic compounds which are similar tonucleotides, but which have an incomplete or abnormal deoxyribose orribose group. Once the nucleotide analogues are in the cell, they arephosphorylated, producing the triphosphate formed which competes withnormal nucleotides for incorporation into the viral DNA or RNA. Once thetriphosphate form of the nucleotide analogue is incorporated into thegrowing nucleic acid chain, it causes irreversible association with theviral polymerase and thus chain termination. Nucleotide analoguesinclude, but are not limited to, acyclovir (used for the treatment ofherpes simplex virus and varicella-zoster virus), gancyclovir (usefulfor the treatment of cytomegalovirus), idoxuridine, ribavirin (usefulfor the treatment of respiratory syncitial virus), dideoxyinosine,dideoxycytidine, zidovudine (azidothymidine), imiquimod, andresimiquimod.

The interferons are cytokines which are secreted by virus-infected cellsas well as immune cells. The interferons function by binding to specificreceptors on cells adjacent to the infected cells, causing the change inthe cell which protects it from infection by the virus, α andβ-interferon also induce the expression of Class I and Class II MHCmolecules on the surface of infected cells, resulting in increasedantigen presentation for host immune cell recognition, α andβ-interferons are available as recombinant forms and have been used forthe treatment of chronic hepatitis B and C infection. At the dosageswhich are effective for anti-viral therapy, interferons have severe sideeffects such as fever, malaise and weight loss.

Anti-viral agents useful in the invention include but are not limited toimmunoglobulins, amantadine, interferons, nucleotide analogues, andprotease inhibitors. Specific examples of anti-virals include but arenot limited to Acemannan; Acyclovir; Acyclovir Sodium; Adefovir;Alovudine; Alvircept Sudotox; Amantadine Hydrochloride; Aranotin;Arildone; Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline;Cytarabine Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine;Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; FamotineHydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet Sodium;Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal;Lamivudine; Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine;Penciclovir; Pirodavir; Ribavirin; Rimantadine Hydrochloride; SaquinavirMesylate; Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine;Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride;Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate; Viroxime;Zalcitabine; Zidovudine; and Zinviroxime.

Anti-fungal agents are useful for the treatment and prevention ofinfective fungi. Anti-fungal agents are sometimes classified by theirmechanism of action. Some antifungal agents function as cell wallinhibitors by inhibiting glucose synthase. These

include, but are not limited to, basiungin/ECB. Other anti-fungal agentsfunction by destabilizing membrane integrity. These include, but are notlimited to, immidazoles, such as clotrimazole, sertaconzole,fluconazole, itraconazole, ketoconazole, miconazole, and voriconacole,as well as FK 463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK292, butenafine, and terbinafine. Other anti-fungal agents function bybreaking down chitin (e.g. chitinase) or immunosuppression (501 cream).

In some embodiments, provided immunomodulatory oligonucleotidecompositions are useful for treating a subject having a cellproliferative disease, such as cancer. A subject having a cancer is asubject that has detectable cancerous cells. The cancer may be amalignant or non-malignant cancer. Cancers or tumors include but are notlimited to biliary tract cancer; brain cancer; breast cancer; cervicalcancer; choriocarcinoma; colon cancer; endometrial cancer; esophagealcancer; gastric cancer; intraepithelial neoplasms; lymphomas; livercancer; lung cancer (e.g. small cell and non-small cell); melanoma;neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostatecancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroidcancer; and renal cancer, as well as other carcinomas and sarcomas. Inone embodiment the cancer is hairy cell leukemia, chronic myelogenousleukemia, cutaneous T-cell leukemia, multiple myeloma, follicularlymphoma, malignant melanoma, squamous cell carcinoma, renal cellcarcinoma, prostate carcinoma, bladder cell carcinoma, or coloncarcinoma.

Provided immunomodulatory oligonucleotide compositions may beadministered alone or in conjunction with an anti-cancer therapy.Anti-cancer therapies include but are not limited to radiation therapy,chemotherapy, immunotherapy, a cancer vaccine, hormone therapy, abiological response modifier, and surgical procedures. A cancermedicament refers to an agent which is administered to a subject for thepurpose of treating a cancer. As used herein, “treating cancer” includespreventing the development of a cancer, reducing the symptoms of cancer,and/or inhibiting the growth of an established cancer. In other aspects,the cancer medicament is administered to a subject at risk of developinga cancer for the purpose of reducing the risk of developing the cancer.Various types of medicaments for the treatment of cancer are describedherein. For the purpose of this specification, cancer medicaments areclassified as chemotherapeutic agents, immunotherapeutic agents, cancervaccines, hormone therapy, and biological response modifiers.

Additionally, the methods of the invention are intended to embrace theuse of more than one cancer medicament along with providedimmunomodulatory oligonucleotide compositions. As an example, whereappropriate, provided immunomodulatory oligonucleotide compositions maybe administered with both a chemotherapeutic agent and animmunotherapeutic agent. Alternatively, the cancer medicament mayembrace an immunotherapeutic agent and a cancer vaccine, or achemotherapeutic agent and a cancer vaccine, or a chemotherapeuticagent, an immunotherapeutic agent and a cancer vaccine all administeredto one subject for the purpose of treating a subject having a cancer orat risk of developing a cancer.

Chemotherapeutic agent may be selected from the group consisting ofmethotrexate, vincristine, adriamycin, cisplatin, non-sugar containingchloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin,doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin,carmustaine and poliferposan, MMI270, BAY 12-9566, RAS famesyltransferase inhibitor, famesyl transferase inhibitor, MMP, MTA/LY231514,LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/Topotecan,PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin,Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, lncel/VX-710,VX-853, ZD0101, ISI641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat,CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317,Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative,Temodal/Temozolomide, Evacet/liposomal doxorubicin, Yewtaxan/Paclitaxel,Taxol/Paclitaxel, Xeload/Capecitabine, Furtulon/Doxifluridine,Cyclopax/oral paclitaxel, Oral Taxoid, SPU-077/Cisplatin, HMR1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/RAS oncogeneinhibitor, BMS-182751/oral platinum, UFT (Tegafur/Uracil),Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer, Campto/Levamisole,Camptosar/lrinotecan, Tumodex/Ralitrexed, Leustatin/Cladribine,Paxex/Paclitaxel, Doxil/liposomal doxorubicin, Caelyx/liposomaldoxorubicin, Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt,ZD1839, LU 79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomaldoxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine seeds,CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide,Ifes/Mesnex/lfosamide, Vumon/Teniposide, Paraplatin/Carboplatin,Plantinol/cisplatin, Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel,prodrug of guanine arabinoside, Taxane Analog, nitrosoureas, alkylatingagents such as melphelan and cyclophosphamide, Aminoglutethimide,Asparaginase, Busulfan, Carboplatin, Chlorambucil, Cytarabine HCI,Dactinomycin, Daunorubicin HCI, Estramustine phosphate sodium, Etoposide(VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolideacetate (LHRH-releasing factor analogue), Lomustine (CCNU),Mechlorethamine HCI (nitrogen mustard), Mercaptopurine, Mesna, Mitotane(o.p′-DDD), Mitoxantrone HCI, Octreotide, Plicamycin, Procarbazine HCI,Streptozocin,

Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine(m-AMSA), Azacitidine, Erthropoietin, Hexamethylmelamine (HMM),Interleukin 2, Mitoguazone (methyl-GAG; methyl glyoxalbis-guanylhydrazone; MGBG), Pentostatin (2′deoxycoformycin), Semustine(methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate, but it is notso limited.

Immunotherapeutic agent may be selected from the group consisting ofRibutaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225,Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior tβ, MDX-210,MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447,MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT,Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5,ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1 D10 Ab,SMART ABL 364 Ab and ImmuRAIT-CEA, but it is not so limited.

Cancer vaccine may be selected from the group consisting of EGF,Anti-idiotypic cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGVganglioside conjugate vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax,STn-KHL theratope, BLP25 (MUC-1), liposomal idiotypic vaccine, Melacine,peptide antigen vaccines, toxin/antigen vaccines, MVA-based vaccine,PACIS, BCG vacine, TA-HPV, TA-CIN, DISC-virus and ImmuCyst/TheraCys, butit is not so limited.

As used herein, the terms “cancer antigen” and “tumor antigen” are usedinterchangeably to refer to antigens which are differentially expressedby cancer cells and can thereby be exploited in order to target cancercells. Cancer antigens are antigens which can potentially stimulateapparently tumor-specific immune responses. Some of these antigens areencoded, although not necessarily expressed, by normal cells. Theseantigens can be characterized as those which are normally silent (i.e.,not expressed) in normal cells, those that are expressed only at certainstages of differentiation and those that are temporally expressed suchas embryonic and fetal antigens. Other cancer antigens are encoded bymutant cellular genes, such as oncogenes (e.g., activated ras oncogene),suppressor genes (e.g., mutant p53), fusion proteins resulting frominternal deletions or chromosomal translocations. Still other cancerantigens can be encoded by viral genes such as those carried on RNA andDNA tumor viruses.

In some embodiments, provided immunomodulatory oligonucleotidecompositions may also be useful for treating and preventing autoimmunedisease. Autoimmune disease is a class of diseases in which a subject'sown antibodies react with host tissue or in which immune effector Tcells are autoreactive to endogenous self peptides and cause destructionof tissue. Thus an immune response is mounted against a subject's ownantigens, referred to as self antigens. Autoimmune diseases include butare not limited to alopecia areata, acquired hemophilia, ankylosingspondylitis, antiphospholipid syndrome, autoimmune-associatedinfertility, autoimmune encephalomyelitis, autoimmune hepatitis,autoimmune hemolytic anemia, autoimmune diabetes mellitus, autoimmunethrombocytopenic purpura, Behget's syndrome, bullous pemphigoid,cardiomyopathy, chronic fatigue immune dysfunction syndrome (CFIDS),chronic inflammatory demyelinating polyneuropathy, Churg-Strausssyndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinindisease, dermatomyositis, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia, fibromyositis, Guillain-Barre syndrome,Hashimoto's thyroiditis, glomerulonephritis (e.g., crescenticglomerulonephritis, proliferative glomerulonephritis), Grave's disease,graft versus host disease, Goodpasture's syndrome, pemphigus (e.g.,pemphigus vulgaris), idiopathic pulmonary fibrosis, idiopathicthrombocytopenic purpura, insulin resistance, idiopathic Addison'sdisease, IgA nephropathy, inflammatory bowel disease (including Crohn'sdisease and ulcerative colitis), juvenile arthritis, lichen planus,myasthenia gravis, multiple sclerosis, mixed connective tissue disease,polymyositis, pernicious anemia, polyarteritis nodosa, polychondritis,polyglandular syndromes, polymyalgia rheumatica, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud'sphenomena, Reite{circumflex over ( )}s syndrome, juvenile and adultrheumatoid arthritis, Sjorgen's syndrome, scleroderma with anti-collagenantibodies, sarcoidosis, stiff-man syndrome, systemic lupuserythematosus (SLE), Takayasu arthritis, transplanted organ rejection,temporal arteritis/giant cell arteritis, uveitis, ulcerative colitis,vasculitis, and vitiligo.

A “self-antigen” as used herein refers to an antigen of a normal hosttissue. Normal host tissue does not include cancer cells. Thus an immuneresponse mounted against a self-antigen, in the context of an autoimmunedisease, is an undesirable immune response and contributes todestruction and damage of normal tissue, whereas an immune responsemounted against a cancer antigen is a desirable immune response andcontributes to the destruction of the tumor or cancer. Thus, in someaspects of the invention aimed at treating autoimmune disorders it isnot recommended that the immunomodulatory oligonucleotides beadministered with self antigens, particularly those that are the targetsof the autoimmune disorder.

In other instances, immunomodulatory oligonucleotides used in accordingto the present invention may be delivered with low doses ofself-antigens. A number of animal studies have demonstrated that mucosaladministration of low doses of antigen can result in a state of immunehyporesponsiveness or “tolerance.” The active mechanism appears to be acytokine-mediated immune deviation away from a Th1 towards apredominantly Th2 and Th3 (i.e., TGF-β dominated) response. The activesuppression with low dose antigen delivery can also suppress anunrelated immune response (bystander suppression) which is ofconsiderable interest in the therapy of autoimmune diseases, forexample, rheumatoid arthritis and SLE. Bystander suppression involvesthe secretion of Th1—counter-regulatory, suppressor cytokines in thelocal environment where proinflammatory and Th1 cytokines are releasedin either an antigen-specific or antigen-nonspecific manner. ‘Tolerance”as used herein is used to refer to this phenomenon. Indeed, oraltolerance has been effective in the treatment of a number of autoimmunediseases in animals including: experimental autoimmune encephalomyelitis(EAE), experimental autoimmune myasthenia gravis, collagen-inducedarthritis (CIA), and insulin-dependent diabetes mellitus. In thesemodels, the prevention and suppression of autoimmune disease isassociated with a shift in antigen-specific humoral and cellularresponses from a Th1 to Th2/Th3 response

Non-limiting examples of useful immunomodulatory oligonucleotides thatcan be utilized in accordance with the present invention are provided inthe accompanying Appendix (B).

RIPtides

In some embodiments, oligonucleotides utilized in accordance with thepresent invention are or act as RNA-interacting polynucleotides(“RIPtides”). RIPtides are a class of therapeutics intended to open upthe space of “undruggable” targets by inactivating structural RNAsimplicated in disease. RIPtides are typically small nucleotide stretches(around 8 nucleotides) that bind and disrupt the action of structuredRNA. Due to their size, these molecules can readily pass through thecell membrane. See, for example, U.S. Pat. No. 6,080,585.

RIPtides may be useful for a number of clinical applications. In thecontext of cancer therapy, at least one target is telomerase, which ispresent in about 90% of cancer cells. Therefore, in some embodiments,stereo-defined oligonucleotides of the invention can be used to targettelomerase for the treatment of various cancer. In addition, RIPtidesmay be useful for the treatment of infectious disease. Viruses, such asHIV and Hepatitis C, must be able to take over a host cell in order toreplicate; therefore, structural RNA can play an important role in thematuration and replication of many viruses. Accordingly, in someembodiments, stereo-defined oligonucleotides of the invention can beused to target portions of DNA or RNA involved in viral maturationand/or replication.

Thus, the invention is useful for treating various viral infections.Viruses include, but are not limited to, enteroviruses (including, butnot limited to, viruses that the family picornaviridae, such as poliovirus, Coxsackie virus, echo virus), rotaviruses, adenovirus, andhepatitis virus, such as hepatitis A, B, C D and E. Specific examples ofviruses that have been found in humans include but are not limited to:Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (alsoreferred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and otherisolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitisA virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g., strains that cause gastroenteritis);Togaviridae (e.g., equine encephalitis viruses, rubella viruses);Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (e.g., coronavi ruses); Rhabdoviridae (e.g.,vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebolaviruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.,influenza viruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovavihdae (papillomaviruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV));Poxviridae (variola viruses, vaccinia viruses, pox viruses);Iridoviridae (e.g., African swine fever virus); and other viruses acutelaryngotracheobronchitis virus, Alphavirus, Kaposi's sarcoma-associatedherpesvirus, Newcastle disease virus, Nipah virus, Norwalk virus,Papillomavirus, parainfluenza virus, avian influenza, SARs virus, WestNile virus.

Antisense Drugs

The present invention also encompasses a variety of antisense-basedtherapeutics. Typically, antisense drugs are small (12-21 nucleotides)DNA- or RNA-like compounds that are chemically modified to engineerfavorable drug properties. Some of the antisense agents available in theart include phosphorothioate or deoxyoligonucleotides, and in thissituation a sulfur group is exchanged for a nonbridging oxygen on thephosphate backbone. This increases the resistance to RNase degradationand improves the pharmacologic stability. Yet in other agents, so-called“the next generation of antisense molecules,” the oligonucleotides the2′-O-methoxyethyl modification to the backbone, which can increase theaffinity for these oligonucleotides for the target RNA. However, in somecases, many of these agents are associated with side effects, such asunwanted immune responses. At least in some situations, such undesirableproperties of these agents are attributable at least in part to theirstereorandom nature. The present invention provides stereodefinedcounterpart having preferred properties, such as improved lipophilicityand increased RNA binding.

A number of antisense-based therapeutics have been developed or indevelopment. Non-limiting examples of antisense therapies are providedbelow. Stereo-defined (e.g., chirally pure) oligonucleotides asdescribed herein can be prepared for achieving improved efficacy,increased affinity to target, reduced side effects, improvedpharmacokinetics, enhanced stability and/or increased bioavailability,relative to the antisense drugs currently available in the art. In someembodiments, antisense therapeutics inclide Morpholino drugs. See, forexample: Morcos, P A (2007). “Achieving targeted and quantifiablealteration of mRNA splicing with Morpholino oligos”. Biochem Biophys ResCommun 358 (2): 521-7.

Cytomegalovirus retinitis: Fomivirsen (marketed as Vitravene), wasapproved by the U.S. FDA in August 1998 as a treatment forcytomegalovirus retinitis.

Hemorrhagic fever viruses: In early 2006, scientists studying the Ebolahemorrhagic fever virus at USAMRIID announced a 75% recovery rate afterinfecting four rhesus monkeys and then treating them with an antisenseMorpholino drug developed by AVI BioPharma, a U.S. biotechnology firm.[U.S. Army Medical Research Institute of Infectious Diseases, FortDetrick, Md. News Release: Gene-Specific Ebola Therapies ProtectNonhuman Primates from Lethal Disease. Jan. 13, 2006] The usualmortality rate for monkeys infected with Ebola virus is 100%. In late2008, AVI BioPharma successfully filed Investigational New Drug (IND)applications with the FDA for its two lead products for Marburg andEbola viruses. These drugs, AVI-6002 (Lu, X; Yu, Q; Binder, GK; Chen, Z;Slepushkina, T; Rossi, J; Dropulic, B (2004). “Antisense-MediatedInhibition of Human Immunodeficiency Virus (HIV) Replication by Use ofan HIV Type 1-Based Vector Results in Severely Attenuated MutantsIncapable of Developing Resistance”. Journal of Virology 78 (13):7079-88) and AVI-6003 are novel analogs based on AVI's PMO antisensechemistry in which anti-viral potency is enhanced by the addition ofpositively-charged components to the morpholino oligomer chain.Preclinical results of AVI-6002 and AVI-6003 demonstrated reproducibleand high rates of survival in non-human primates challenged with alethal infection of the Ebola and Marburg viruses, respectively (MedicalNews Today. AVI BioPharma Announces FDA Clears IND Applications ForClinical Trials Of RNA Therapeutic Agents For Treatment Of Ebola AndMarburg Viruses. 30 Dec. 2008).

Cancer: Also in 2006, German physicians reported on a dose-escalationstudy for the compound AP 12009 (a phosphorothioate antisenseoligodeoxynucleotide specific for the mRNA of human transforming growthfactor TGF-beta2) in patients with high grade gliomas. At the time ofthe report, the median overall survival had not been obtained and theauthors hinted at a potential cure (Results of G0004, a phase IIbactively controlled clinical trial with the TGF-b2 targeted compound AP12009 for recurrent anaplastic astrocytoma—Hau et al. 24 (18Supplement): 1566—ASCO Meeting Abstracts).

For example, trabedersen (AP 12009-P001) is developed as monotherapy forthe treatment of patients with advanced pancreatic cancer, malignantmelanoma, brain tumors, anaplastic astrocytoma, and colorectalcarcinoma. AP 12009-P001 has been evaluated in an open-label,multicenter, Phase I/II dose escalation trial to evaluate safety andtolerability of i.v. administration of trabedersen in 61 patients withadvanced solid tumors known to overproduce TGF-b2, who were either notor no longer amenable to established forms of therapies.

Other examples of antisense drugs that target proteins overexpressed incancers include: Clusterin (OGX-011/TV01011), which regulatesepithelial-mesenchymal transition induced by the growth factor TGF betaand the transcription factor TWIST1 in prostate cancer cells; ATL1101 (asecond generation antisense inhibitor of the insulin-like growthfactor-I receptor (IGF-IR)) in prostate cancer treatment; oblimersen(trade name: Genasense) for treating chronic lymphocytic leukemia; G4460and LR3001 (inhibitors of c-myb) for the treatment of CML, melanoma,neuroblastoma, and cancers of the breast, pancreas and colon; MG98(inhibitor of DNA methyltransferase I) for the treatment of renal cellcarcinoma; ISIS 5132 (c-Raf antisense); LY900003 (Protein kinase C-alphaantisense); G3139 (Bcl-2 antisense), etc.

HIV/AIDS: Starting in 2004, researchers in the US have been conductingresearch on using antisense technology to combat HIV. It is known thatantisense-mediated inhibition of human immunodeficiency virus (HIV)replication by use of an HIV type 1-based vector results in severelyattenuated mutants incapable of developing resistance. In February 2010researchers reported success in reducing HIV viral load using patientT-cells which had been harvested, modified with an RNA antisense strandto the HIV viral envelope protein, and re-infused into the patientduring a planned lapse in retroviral drug therapy.

High cholesterol: In 2010 mipomersen (previously ISIS 301012, trade nameKynamro) successfully completed phase 3 trials for some types of highcholesterol. Mipomersen is a cholesterol-reducing drug candidate. It isan antisense therapeutic that targets the messenger RNA forapolipoprotein B. See: Merki E, Graham M J, Mullick A E, et al. (August2008). “Antisense oligonucleotide directed to human apolipoprotein B-100reduces lipoprotein(a) levels and oxidized phospholipids on humanapolipoprotein B-100 particles in lipoprotein(a) transgenic mice”.Circulation 118 (7): 743-53; El Harchaoui K, Akdim F, Stroes E S, Trip MD, Kastelein J J (2008). “Current and future pharmacologic options forthe management of patients unable to achieve low-densitylipoprotein-cholesterol goals with statins”. Am J Cardiovasc Drugs 8(4): 233-42; Athyros V G, Kakafika A I, Tziomalos K, Karagiannis A,Mikhailidis D P (July 2008). “Antisense technology for the prevention orthe treatment of cardiovascular disease: the next blockbuster?”. ExpertOpin Investig Drugs 17 (7): 969-72. It is administered as a weeklyinjection. The compound is a ‘second-generation’ antisenseoligonucleotide; the nucleotides are linked with phosphorothioatelinkages rather than the phosphodiester linkages of RNA and DNA, and thesugar parts are deoxyribose in the middle part of the molecule and2′-O-methoxyethyl-modified ribose at the two ends. These modificationsmake the drug resistant to degradation by nucleases, allowing it to beadministered weekly. The drug accumulates in the liver, which isconvenient since apolipoprotein B predominantly acts there. The completesequence isG*-C*-C*-U*-C*-dA-dG-dT-dC-dT-dG-dmC-dT-dT-dmC-G*-C*-A*-C*-C* (SEQ IDNO: 113) [d=2′-deoxy, *=2′-O-(2-methoxyethyl)] with 3′-5′phosphorothioate linkages. The Phase 3 trials were in patients withfamilial hypercholesterolemia (FH), both homozygous (ho) andheterozygous (he). FH is a genetic disorder that causes exceptionallyhigh levels of low-density lipoprotein cholesterol. Both trials showedexceptional performance with the highest efficacy seen so far in thosetwo patient populations, and with relatively low drop-out rates comparedto other injectable drugs.

Duchenne muscular dystrophy: In Duchenne muscular dystrophy (DMD), thepatient's muscle cells break down and are lost, leading to progressivemuscle weakness and death. AVI-4658 is a targeted antisense therapy torestore expression of dystrophin, a key protein which patients withDuchenne muscular dystrophy lack. Results from an open-label, phase 2,dose-escalation study with 19 patients showed that when treatment wascompleted, a muscle biopsy was taken from each patient. The teamdiscovered that the patients' ability to produce functional mRNA through“exon skipping’ was repaired with the use of AVI-4658, eventuallyallowing them to manufacture functional dystrophin protein.

Myotonic dystrophy: Myotonic dystrophy is the most common musculardisease in adults, affecting mainly the skeletal muscles, heart andcentral nervous system. It occurs because of a mutation that causesnumerous repeats of three letters of the genetic code (CTG) in a genecalled DMPK. The messenger RNA that is produced from the mutated genealso contains the abnormal long repeats that cause the RNA to accumulatein the cell's nucleus. There it sequesters and blocks the function of aprotein called Muscleblind-like 1 and activates another protein calledCELF1. These proteins antagonize one another and the result is abnormalexpression of proteins from many other genes in adult tissues, resultingin disease. To counteract this, Cooper and his colleagues createdantisense oligonucleotides that are simply strands of genetic materialthat seek out portions of the abnormal RNA repeats and target RNase H tothe toxic RNA causing its degradation. It was also reported thatcombining the antisense oligonucleotides with other antisenseoligonucleotides that help released the sequestered Muscleblind-like1can enhance the effect.

Diabetes: SGLT2 (ISIS 388626) is an antisense drug to achieve reductionin sodium dependent glucose co-transporter type 2 (SGLT2) levels thatresulted in a significant reduction in blood glucose levels in diabeticpatients.

Hepatitis: Persistent hepatitis C virus (HCV) infection is a leadingcause of liver disease. Antisense oligonucleotides represent a promisingclass of antiviral agents. Such drugs under development include ISIS14803, which is a 20-unit phosphorothioate oligodeoxynucleotide thatinhibits HCV replication and protein expression.

Methods of Treatment

Provided oligonucleotides and compositions thereof, described herein,are useful as therapeutic agents against various disease states,including use as antiviral agents. In some embodiments, providedoligonucleotides can be used as agents for treatment of diseases throughmodulation of DNA and/or RNA activity. In some embodiments, providedoligonucleotides can be used for inhibiting specific gene expression.For example, a provided oligonucleotide can be complementary to aspecific target messenger RNA (mRNA) sequence. It can be used to inhibitviral replication of myriad viruses. Exemplary virus families includeorthomyxoviruses, pox viruses, herpes viruses, papilloma viruses,picornaviruses, flaviviruses, retroviruses, hepatitis viruses,paramyxoviruses, reoviruses, parvoviruses, filoviruses, coronaviruses,arenaviruses, rhabdoviruses and adenoviruses. Additional virus familiesare known and are also contemplated herein. Other examples include usesas antisense compounds against HIV RNA or other retroviral RNA or forhybridizing to HIV mRNA encoding the tat protein, or to the TAR regionof HIV mRNA. In some embodiments, the nucleic acids mimic the secondarystructure of the TAR region of HIV mRNA, and by doing so bind the tatprotein. In some embodiments, a provided oligonucleotide is used toinhibit expression of a target protein by contacting a cell with aprovided oligonucleotide, wherein the expression of other proteins inthe cell are not inhibited or are minimally inhibited. In someembodiment, target protein inhibition occurs in vivo in a mammal. Insome embodiments, a therapeutically effective amount of a providedoligonucleotide is administered for inhibiting the expression of atarget protein.

Other examples of proteins where expression can be modulated include JunN-terminal kinase (JNK) proteins, diacylglycerol acyltransferase I,apolipoprotein B, glucagon receptor, Aurora B, acyl CoA cholesterolacyltransferase-2, c-reactive protein, STAT (signal transducers andactivators of transcription) family of proteins, and MDR P-glycoprotein.In some embodiments, a provided oligonucleotide can be used to inhibitprotein phosphatase 1B (PTP1B) expression, RNA-dependent RNA viralpolymerase. In some embodiments, a provided oligonucleotide can be usedto induce events such as apoptosis in cancer cells or to make a cellmore susceptible to apoptosis. In some embodiments, a providedoligonucleotide can be used to modulate activities of proteins. In someembodiments, a provided oligonucleotide can help modulate RNase Hactivity targeting multidrug resistance (MDR) RNA molecules.

In some embodiments, the present invention provides methods of treatinga disease mediated by undesired gene expression in a subject (e.g.,mammals, such as humans) in need of such treatment. By “diseases” ismeant diseases, or disease symptoms. Methods include, e.g.,administering to the subject an effective amount of a providedoligonucleotide.

Examples of diseases mediated by undesired gene expression includecancer (e.g., leukemia, tumors, and metastases), allergy, asthma,obesity, inflammation (e.g., inflammatory diseases such as inflammatoryairways disease), hypercholesterolemia, hematological disorders, severeacute respiratory syndrome (SARS), obstructive airways disease, asthma,autoimmune diseases, retroviral diseases such as AIDS or HIV, otherviral infections, intrauterine infections, metabolic diseases, infection(e.g., bacterial, viral, yeast, fungal), CNS diseases, brain tumors,degenerative neural diseases, cardiovascular diseases, and diseasesassociated with angiogenesis, neovascularization, and vasculogenesis.

In some embodiments, provided oligonucleotides are useful for treatingcancer, including pancreatic cancer, and other diseases or disordersinvolving abnormal cell proliferation.

Located in the upper abdomen (in the retroperitoneum), the pancreas is adual-function gland of the digestive and endocrine system. In certaininstances, the pancreas functions as an endocrine gland (e.g., producingseveral important hormones). In certain instances, the pancreasfunctions as an exocrine gland (e.g., secreting fluids containingdigestive enzymes that pass to the small intestine).

Pancreatic cancer is the fourth most common cause of cancer death in theUS (after lung, colon and breast), comprising 6% of all cancer-relateddeaths. In 2008, an estimated 37,680 new cases of pancreatic cancer willhave been diagnosed in the US, with 34,290 deaths. Incidence of thedisease, rises linearly after age 50, with the only definitive riskfactor being cigarette smoking (smokers are four times more likely todevelop the disease than non-smokers). Invasive pancreatic cancer isalmost always fatal. The collective median survival time of all patientsis 4-6 months. Relative 1-year survival is 24%; the overall 5-yearsurvival rate <5%.

Pancreatic cancer is asymptomatic in its early stage and often remainsundiagnosed for several months (less than one third of patients beingdiagnosed within 2 months of the onset symptoms). In certain instances,the delayed diagnosis results in (either partially or fully) metastasisof the cancerous cells to the liver or lymph nodes.

Currently, surgery (resectioning of the pancreas) is the primary andonly curative therapy for pancreatic cancer. However, only 15-25% oftumors are resectable at the time of diagnosis and only 10-20% ofpatients undergoing surgery survive more than two years. Once tumorinfiltration occurs and other tissues have been affected, surgery is nolonger possible.

In certain instances, diabetes mellitus or pancreatitis predisposes anindividual to develop a proliferative disorder of a plurality ofpancreatic cells. In certain instances, individuals are at an increasedrisk of developing a proliferative disorder of a plurality of pancreaticcells due to a hereditary syndrome selected from the group consisting ofhereditary nonpolyposis colorectal cancer (HNPCC) and familialadenomatous polyposis (FAP). In certain instances, individuals are at anincreased risk of developing a proliferative disorder of a plurality ofpancreatic cells due to a mutation in a gene selected from the groupconsisting of MSH2, MSH6, MLH1, and APC.

Ideally, effective treatment of pancreatic cancer should (i) control theprimary tumor mass, both initially and subsequently, and (ii) treat themetastatic tumor cells. Chemoprevention (the administration of agentssuch as drugs, biologics, nutrients and the like) slows the progressionof, reverses, or inhibits carcinogenesis, thereby lowering the risk ofdeveloping invasive or clinically significant disease.

Disclosed herein, in certain embodiments, are methods of treatingpancreatic cancer. As used herein, “pancreatic cancer” includes forms ofcancer of the pancreas. In some embodiments, a pancreatic cancer ismetastatic pancreatic cancer. In some embodiments, a pancreatic canceris a carcinoma, sarcoma, cancer, or combinations thereof. In someembodiments, a pancreatic cancer to be treated includes sporadic andhereditary pancreatic cancers. In some embodiments, a pancreatic canceris duct cell carcinoma, acinar cell carcinoma, papillary mucinouscarcinoma, signet ring carcinoma, adenosquamous carcinoma,undifferentiated carcinoma, mucinous carcinoma, giant cell carcinoma,small cell carcinoma, cystcancer, serous cystcancer, mucinouscystcancer, unclassified pancreatic cancer, pancreatoblastoma, orcombinations thereof.

In some embodiments, an individual in need of treatment for pancreaticcancer presents with a localized tumor of the pancreas. In someembodiments, an individual in need of treatment for pancreatic cancerpresents with a negative regional lymph node biopsy. In someembodiments, an individual in need of treatment for pancreatic cancerpresents with a positive regional lymph node biopsy. In someembodiments, an individual in need of treatment for pancreatic cancerpresents with a nodal negative pancreatic tumor (e.g., node-negative).In some embodiments, an individual in need of treatment for pancreaticcancer presents with a nodal positive tumor (e.g., node-positive).

In some embodiments, a pancreatic cancer in an individual in need oftreatment for pancreatic cancer has metastasized to other locations inthe body. In some embodiments, a pancreatic cancer has metastasized to alocation selected from the group consisting of lymph node, stomach, bileduct, liver, bone, ovary, peritoneum and brain.

In some embodiments, cancer cells or precancerous cells are identifiedby histological typing or grading of a tissue sample (e.g., a biopsysample). In some embodiments, cancer cells or precancerous cells areidentified through the use of appropriate molecular markers.

In some embodiments, a pancreatic cancer in an individual in need oftreatment for pancreatic cancer is staged according to the AmericanJoint Committee on Cancer (AJCC) TNM classification system, where thetumor (T) has been assigned a stage of Tx, T1, T2, T3, T4; and where theregional lymph nodes (N) have been assigned a stage of NX, N0, N1; andwhere distant metastasis (M) has been assigned a stage of MX, M0, or M1.In some embodiments, a pancreatic cancer in an individual in need oftreatment for pancreatic cancer is staged as Stage 0, I, IA, IB, II IIA,IIB, III, and IV pancreatic cancer. In some embodiments, a pancreaticcancer in an individual in need of treatment for pancreatic cancer isstaged as Grade GX (e.g., grade cannot be assessed), Grade 1, Grade 2,Grade 3 or Grade 4.

More specific examples of cancers treated with provided oligonucleotidesinclude breast cancer, lung cancer, melanoma, colorectal cancer, bladdercancer, ovarian cancer, prostate cancer, renal cancer, squamous cellcancer, glioblastoma, Kaposi's sarcoma, multiple myeloma, and leukemia.

Evaluation and Treatment of Cancer

The term “tumor cell antigen” is defined herein as an antigen that ispresent in higher quantities on a tumor cell or in body fluids thanunrelated tumor cells, normal cells, or in normal body fluid. Theantigen presence may be tested by any number of assays known to thoseskilled in the art and include without limitation negative and/orpositive selection with antibodies, such as an ELISA assay, aradioimmunoassay, or by Western Blot.

“Apoptosis inducing agent” is defined herein to induceapoptosis/programmed cell death, and include, for example, anticanceragents and treatments wherein cells (e.g., tumor cells) are induced toundergo programmed cell death. Exemplary apoptosis inducing agents aredescribed in more detail below.

The terms “apoptosis” or “programmed cell death,” refers to thephysiological process by which unwanted or useless cells are eliminatedduring development and other normal biological processes. Apoptosis is amode of cell death that occurs under normal physiological conditions andthe cell is an active participant in its own demise (“cellularsuicide”). It is most often found during normal cell turnover and tissuehomeostasis, embryogenesis, induction and maintenance of immunetolerance, development of the nervous system and endocrine-dependenttissue atrophy. Cells undergoing apoptosis show characteristicmorphological and biochemical features. These features include chromatinaggregation, nuclear and cytoplasmic condensation, partition ofcytoplasm and nucleus into membrane bound vesicles (apoptotic bodies),which contain ribosomes, morphologically intact mitochondria and nuclearmaterial. In vivo, these apoptotic bodies are rapidly recognized andphagocytized by macrophages, dendritic cells or adjacent epithelialcells. Due to this efficient mechanism for the removal of apoptoticcells in vivo no inflammatory response is elicited. In vitro, theapoptotic bodies as well as the remaining cell fragments ultimatelyswell and finally lyse. This terminal phase of in vitro cell death hasbeen termed “secondary necrosis.” Apoptosis can be measured by methodsknown to those skilled in the art like DNA fragmentation, exposure ofAnnexin V, activation of caspases, release of cytochrome c, etc. A cellthat has been induced to die is termed herein as an “apoptotic cell.”Apoptosis can also be tested using a standard Annexin V Apoptosis Assay:NIH:OVCAR-3 cells are grown in 6-well plates (NUNC) and irradiated ortreated with an antagonist (or in combination with another anti-cancerdrug) for 4-48 hours, washed and stained with Annexin V-FITC(BD-Pharmingen) for 1 hour. Cells are analyzed by flow cytometry(Becton-Dickinson, CellQuest), counterstained with Propidium Iodide andanalyzed again in the flow cytometer.

Patients can be assessed with respect to symptoms at one or moremultiple time points including prior to, during, and after treatmentregimens. Treatment can result in improving the subject's condition andcan be assessed by determining if one or more of the following factorshas occurred: decreased tumor size, decreased cell proliferation,decreased numbers of cells, decreased neovascularization, increasedapoptosis, or decreased survival of at least a portion of the tumorcells. One or more of these occurrences may, in some cases, result inpartial or total elimination of the cancer and prolongation of survivalof the patient. Alternatively, for terminal stage cancers, treatment mayresult in stasis of disease, better quality of life and/or prolongationof survival.

Methods of Assaying Cell Migration

Assays for cell migration have been described in the literature, e.g.,by Brooks, et al., J. Clin. Invest 1997, 99:1390-1398 and methods formeasuring cell migration are known to those of skill in the art. In onemethod for measuring cell migration described herein, membranes fromtranswell migration chambers are coated with substrate, the transwellswashed, and non-specific binding sites blocked with BSA. Tumor cellsfrom sub-confluent cultures are harvested, washed, and resuspended inmigration buffer in the presence or absence of assay antibodies. Afterthe tumor cells are allowed to migrate to the underside of the coatedtranswell membranes, the cells remaining on the top-side of the membraneare removed and cells that migrate to the under-side are stained withcrystal violet. Cell migration is then quantified by direct cell countsper microscopic field.

Methods of Assaying Tumor Growth

Tumor growth can be assayed by methods known to those of skill in theart, e.g., the SCID mouse model, the nude mouse model, and BALB/c micewith syngeneic tumors. SCID mouse models for tumor growth are carriedout as follows: subconfluent human M21 melanoma cells (or any desiredtumor cell type) are harvested, washed, and resuspended in sterile PBS(20×106 per mL). SCID mice are injected subcutaneously with 100 L of M21human melanoma cell (2×106) suspension. Three days after tumor cellinjection, mice are either untreated or treated intraperitoneally withan antagonist in the desired dose ranges. The mice are treated daily for24 days. Tumor size is measured with calipers and the volume estimatedusing the formula V=(L×W2)/2, where V is equal to the volume, L is equalto the length, and W is equal to the width.

Alternatively, nude mouse models, SCID mouse models and/or BALB/csyngeneic mouse models can also be utilized to assess tumor growth andinhibition thereof by the humanized anti-endoglin antibodies orantigen-binding fragments described herein.

Methods of Assaying Cell Proliferation

Cell proliferation can be assayed by methods known to those of skill inthe art. As described herein, subconfluent human endothelial cells(HUVECs) can be resuspended in proliferation buffer containing low(5.0%) serum in the presence or absence of CM (25 μL) from ECV or ECVLcells, and endothelial cells allowed to proliferate for 24 hours.Proliferation can be quantified by measuring mitochondrial dehydrogenaseactivity using a commercially available WST-1 assay kit (Chemicon).Also, as described herein, proliferation can be quantified by measuring3H incorporation using standard methods. (She et al., Int. J. Cancer,108: 251-257 (2004)).

Other methods of assessing cell proliferation are known in the art andare contemplated herein. Further non-limiting examples are described inmore detail in the examples.

One would understand that classification and staging systems describedherein represent one means to assess treatment of cancers describedherein; additionally, other staging schemes are known in the art and maybe used in connection with the methods described herein. By way ofexample only, the TNM classification of malignant tumors may be used asa cancer staging system to describe the extent of cancer in a patient'sbody. T describes the size of the tumor and whether it has invadednearby tissue, N describes regional lymph nodes that are involved, and Mdescribes distant metastasis. TNM is maintained by the InternationalUnion Against Cancer (UICC) and is used by the American Joint Committeeon Cancer (AJCC) and the International Federation of Gynecology andObstetrics (FIGO). One would understand that not all tumors have TNMclassifications such as, for example, brain tumors. Generally, T(a,is,(0), 1-4) is measured as the size or direct extent of the primarytumor. N (0-3) refers to the degree of spread to regional lymph nodes:N0 means that tumor cells are absent from regional lymph nodes, N1 meansthat tumor cells spread to the closest or small numbers of regionallymph nodes, N2 means that tumor cells spread to an extent between N1and N3; N3 means that tumor cells spread to most distant or numerousregional lymph nodes. M (0/1) refers to the presence of metastasis: M0means that no distant metastasis are present; M1 means that metastasishas occurred to distant organs (beyond regional lymph nodes). Otherparameters may also be assessed. G (1-4) refers to the grade of cancercells (i.e., they are low grade if they appear similar to normal cells,and high grade if they appear poorly differentiated). R (0/1/2) refersto the completeness of an operation (i.e., resection-boundaries free ofcancer cells or not). L (0/1) refers to invasion into lymphatic vessels.V (0/1) refers to invasion into vein. C (1-4) refers to a modifier ofthe certainty (quality) of V.

Provided herein are methods for degrading, inhibiting the growth of orkilling cancer cells comprising contacting the cells with an amount of acompound described herein effective to degrade, inhibit the growth of orkill cancer cells.

Provided herein are methods of inhibiting tumor size increase, reducingthe size of a tumor, reducing tumor proliferation or preventing tumorproliferation in an individual comprising administering to saidindividual an effective amount of a compound described herein to inhibittumor size increase, reduce the size of a tumor, reduce tumorproliferation or prevent tumor proliferation. Treatment of tumors insome cases includes stasis of symptoms, that is, by treating thepatient, the cancer does not worsen and survival of the patient isprolonged.

Patients may be assessed with respect to symptoms at one or moremultiple time points including prior to, during, and after treatmentregimens. Treatment can result in improving the subject's condition andcan be assessed by determining if one or more of the following eventshas occurred: decreased tumor size, decreased tumor cell proliferation,decreased numbers of cells, decreased neovascularization and/orincreased apoptosis. One or more of these occurrences may, in somecases, result in partial or total elimination of the cancer andprolongation of survival of the patient. Alternatively, for terminalstage cancers, treatment may result in stasis of disease, better qualityof life and/or prolongation of survival.

Other methods of assessing treatment are known in the art andcontemplated herein. In an exemplary embodiment, the pro-oligonucleotidecompounds of the invention are administered to a subject such as amammal (e.g., a human), suffering from a medical disorder, e.g., acancer, or non-malignant conditions characterized by the presence of aclass of unwanted cells.

Primary outcome measures may be assessed for patients treated using themethods described herein and include, for example, progression-freesurvival. In one embodiment, an increase in progression free survival isobserved in an amount of by about 2-fold, 5-fold, 10-fold, 20 fold, 50fold or more compared to lack of treatment. In another embodiment, anincrease in progression free survival is increased survival by about 3months, about 6 months, about 9 months, about 12 months, about 18months, about 2 years, about 3 years, about 4 years, about 5 years ormore compared to lack of treatment.

Secondary outcome measures may also be assessed and include duration ofresponse, time to tumor progression, overall survival, serious andnon-serious adverse events. For example, a treatment may preventprogression of the disease (i.e., stasis) or may result in animprovement. Alternately, or in addition, other goals can be measuredwith respect to one or more of the following: decreased tumor burden,decreased neovascularization, reduced side effects, decreased adversereactions, and/or increased patient compliance.

Other specific examples of diseases or disorders for which treatment bythe compounds or compositions of the invention are useful for treatmentor prevention include, but are not limited to transplant rejection(e.g., kidney, liver, heart, lung, islet cells, pancreas, bone marrow,cornea, small bowel, skin allografts or xenografts and othertransplants), graft vs. host disease, osteoarthritis, rheumatoidarthritis, multiple sclerosis, diabetes, diabetic retinopathy,inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis,and other bowel diseases), renal disease, cachexia, septic shock, lupus,myasthenia gravis, psoriasis, dermatitis, eczema, seborrhea, Alzheimer'sdisease, Parkinson's disease, stem cell protection during chemotherapy,ex vivo selection or ex vivo purging for autologous or allogeneic bonemarrow transplantation, ocular disease, retinopathies (e.g., maculardegeneration, diabetic retinopathy, and other retinopathies), cornealdisease, glaucoma, infections (e.g., bacterial, viral, or fungal), heartdisease, including, but not limited to, restenosis.

Activation of RNAse L

The 2′-5′ oligoadenylate (2-5A)/RNase L pathway is one of the enzymaticpathways induced by interferon. Rnase L is activated after binding to5′-phosphorylated fragments of 2′-5′ adenylic acid. These fragments of2′-5′ adenylic acid (2-5A) are produced under the control of 2′-5′oligo(A) synthetase. This pathway is part of the innate immune systemand has an important role in preventing viral infection. 2-5A-Inducedcleavage of single-stranded RNA results in apoptosis. Biostablephosphorothioate analogs of 2-5A have been shown to be potent activatorsof Rnase L (Xianh et al., Cancer Research (2003), 63:6795-6801). In thisstudy, the 2-5A analogs induced Rnase L activity and caused apoptosis incultures of late-stage, metastatic human prostate cancer cell linesDU145, PC3 and LNCaP.

Sustained activation of RNase L triggers a mitochondrial pathway ofapoptosis that eliminates virus-infected cells as well ascancerous/tumor cells. RNase L can inhibit fibrosarcoma growth, prostatecancer growth, colorectal cancer growth and pancreatic cancer growth.Given the common role of RNase L in different cancers, it iscontemplated that the invention described herein can be use for thetreatment of any type of cancer. Silverman, R H, Cytokine Growth FactorRev, 18(5-6): 381-388 (2007); Bisbal, C. and Silverman, R H, Biochimie.89(6-7): 789-798 (2007). By way of example, downregulation of RNase Lrefers to any reduction in expression levels of the gene or genesencoding RNase L, silencing of the gene or genes encoding RNase L,reduction in the levels of expression/translation of the proteinscomprising RNase L, reduction in the amount of RNase L present within acell, and/or any reduction in activity of RNase L as compared to apredetermined level of RNase L in an exemplary healthy population.Alternatively any reduction in RNase L levels as described herein can beindicative of downregulation of RNase L.

In some embodiments, provided oligonucleotides are useful for thetreatment of diseases having downregulated RNase L. In some embodiment,a disease associated with downregulated RNase L is cancer. In someembodiments, the cancer is pancreatic cancer, prostate cancer, orcolorectal cancer. Alternatively, provided oligonucleotides describedherein are useful for the treatment of disease having upregulated RNaseL. In some embodiments, the disease having upregulated RNase L ischronic fatigue syndrome. Additional diseases having upregulated RNase Lare known in the art and contemplated herein.

When used as therapeutics, a provided oligonucleotide is administered asa pharmaceutical composition. In some embodiments, the pharmaceuticalcomposition comprises a therapeutically effective amount of a providedoligonucleotide comprising, or a pharmaceutically acceptable saltthereof, and at least one pharmaceutically acceptable inactiveingredient selected from pharmaceutically acceptable diluents,pharmaceutically acceptable excipients, and pharmaceutically acceptablecarriers. In another embodiment, the pharmaceutical composition isformulated for intravenous injection, oral administration, buccaladministration, inhalation, nasal administration, topicaladministration, ophthalmic administration or otic administration. Infurther embodiments, the pharmaceutical composition is a tablet, a pill,a capsule, a liquid, an inhalant, a nasal spray solution, a suppository,a suspension, a gel, a colloid, a dispersion, a suspension, a solution,an emulsion, an ointment, a lotion, an eye drop or an ear drop.

Pharmaceutical Compositions

When used as therapeutics, a provided oligonucleotide or oligonucleotidecomposition described herein is administered as a pharmaceuticalcomposition. In some embodiments, the pharmaceutical compositioncomprises a therapeutically effective amount of a providedoligonucleotides, or a pharmaceutically acceptable salt thereof, and atleast one pharmaceutically acceptable inactive ingredient selected frompharmaceutically acceptable diluents, pharmaceutically acceptableexcipients, and pharmaceutically acceptable carriers. In someembodiments, the pharmaceutical composition is formulated forintravenous injection, oral administration, buccal administration,inhalation, nasal administration, topical administration, ophthalmicadministration or otic administration. In some embodiments, thepharmaceutical composition is a tablet, a pill, a capsule, a liquid, aninhalant, a nasal spray solution, a suppository, a suspension, a gel, acolloid, a dispersion, a suspension, a solution, an emulsion, anointment, a lotion, an eye drop or an ear drop.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising chirally controlled oligonucleotide, orcomposition thereof, in admixture with a pharmaceutically acceptableexcipient. One of skill in the art will recognize that thepharmaceutical compositions include the pharmaceutically acceptablesalts of the chirally controlled oligonucleotide, or compositionthereof, described above.

Compounds for Enhancing and Targeting Delivery

A provided oligonucleotide as described herein can be delivered using avariety of delivery strategies, including conjugates of nucleic acidswith various ligands, as well as use of nanocarrier approaches. Anynucleic acid delivery strategies are contemplated for use with providedoligonucleotides described herein. The choice between exemplary deliverystrategies, including but not limited to, chemical conjugates, cationiclipid/liposomal transfer vessicles and supramolecular nanocarriersdepends on the therapeutic context, and methods for determining theoptimal delivery modality are known in the art and further contemplatedherein.

Cell Penetrating Compounds (“CPCs”)

Numerous compounds are known to act as carriers for cargo such asnucleic acids and facilitate entry of the nucleic acid in a cell in anin vivo setting. Exemplary carriers are described in Dietz et al.,Molecular & Cellular Neuroscience, 27(2): 85-131 (2004) which isincorporated herein by reference. The prototypical CPCs derived from theTat and antennepedia transcriptional regulators have been joined by alarge number of new moieties. As an example, CPCs that are peptides canbe relatively short (9-30 amino acids) polycationic peptides rich inargine and lysine, or membrane-interactive hydrophobic sequences. CPCscan be linked by recombinant DNA techniques or chemically coupled topeptides, oligonucleotides or nanocarriers, which then comprise the‘cargo’ for the CPC.

Cell Targeting Ligands (“CTLs”)

Another strategy is to deliver oligonucleotides by use of a CTL thatbinds with high affinity to a cell surface receptor that is capable ofundergoing efficient internalization. Potential ligands includeantibodies, polypeptides derived from phage display libraries, and smallorganic molecules. Additional cell-targeting ligands are known in theart, or will be developed, and are contemplated for use with theinvention described herein (for ASGPR—GalNAc conjugated siRNA andoligonucleotides, e.g., WO2012037254A1). Because various receptors areoften preferentially expressed on particular cell types, this approachoffers the possibility of improved selectivity for the oligonucleotidereagents. Exemplary receptor targets include, but are not limited to,lipoprotein receptors (such as those in the liver), integrins, receptortyrosine kinases, and the G-protein coupled receptor (GPCR) superfamily.

Nanocarriers

A variety of supramolecular nanocarriers can be used to deliver nucleicacids. Exemplary nanocarriers include, but are not limited to liposomes,cationic polymer complexes and various polymeric. Complexation ofnucleic acids with various polycations is another approach forintracellular delivery; this includes use of PEGlyated polycations,polyethyleneamine (PEI) complexes, cationic block co-polymers, anddendrimers. Several cationic nanocarriers, including PEI andpolyamidoamine dendrimers help to release contents from endosomes. Otherapproaches include use of polymeric nanoparticles, polymer micelles,quantum dots and lipoplexes.

Additional nucleic acid delivery strategies are known in addition to theexemplary delivery strategies described herein.

In therapeutic and/or diagnostic applications, the compounds of theinvention can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington, The Science andPractice of Pharmacy, (20th ed. 2000).

Provided oligonucleotides, and compositions thereof, are effective overa wide dosage range. For example, in the treatment of adult humans,dosages from about 0.01 to about 1000 mg, from about 0.5 to about 100mg, from about 1 to about 50 mg per day, and from about 5 to about 100mg per day are examples of dosages that may be used. The exact dosagewill depend upon the route of administration, the form in which thecompound is administered, the subject to be treated, the body weight ofthe subject to be treated, and the preference and experience of theattending physician.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art, and may include, by way of example but notlimitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington, The Science and Practice of Pharmacy (20th ed. 2000).Preferred pharmaceutically acceptable salts include, for example,acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide,hydrochloride, maleate, mesylate, napsylate, pamoate (embonate),phosphate, salicylate, succinate, sulfate, or tartrate.

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained-low release form as is known to those skilled in theart. Techniques for formulation and administration may be found inRemington, The Science and Practice of Pharmacy (20th ed. 2000).Suitable routes may include oral, buccal, by inhalation spray,sublingual, rectal, transdermal, vaginal, transmucosal, nasal orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intra-articullar, intra-sternal,intra-synovial, intra-hepatic, intralesional, intracranial,intraperitoneal, intranasal, or intraocular injections or other modes ofdelivery.

For injection, the agents of the invention may be formulated and dilutedin aqueous solutions, such as in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.For such transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the invention intodosages suitable for systemic administration is within the scope of theinvention. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present invention, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection.

The compounds can be formulated readily using pharmaceuticallyacceptable carriers well known in the art into dosages suitable for oraladministration. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a subject(e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the invention may alsobe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

Depending upon the particular condition, or disease state, to be treatedor prevented, additional therapeutic agents, which are normallyadministered to treat or prevent that condition, may be administeredtogether with the inhibitors of this invention. For example,chemotherapeutic agents or other anti-proliferative agents may becombined with the inhibitors of this invention to treat proliferativediseases and cancer. Examples of known chemotherapeutic agents include,but are not limited to, adriamycin, dexamethasone, vincristine,cyclophosphamide, fluorouracil, topotecan, taxol, interferons, andplatinum derivatives.

Other examples of agents the non-racemic pro-oligonucleotide of thisinvention may also be combined with include, without limitation,anti-inflammatory agents such as corticosteroids, TNF blockers, IL-1 RA,azathioprine, cyclophosphamide, and sulfasalazine; immunomodulatory andimmunosuppressive agents such as cyclosporin, tacrolimus, rapamycin,mycophenolate mofetil, interferons, corticosteroids, cyclophophamide,azathioprine, and sulfasalazine; neurotrophic factors such asacetylcholinesterase inhibitors, MAO inhibitors, interferons,anti-convulsants, ion channel blockers, riluzole, and anti-Parkinsonianagents; agents for treating cardiovascular disease such asbeta-blockers, ACE inhibitors, diuretics, nitrates, calcium channelblockers, and statins; agents for treating liver disease such ascorticosteroids, cholestyramine, interferons, and anti-viral agents;agents for treating blood disorders such as corticosteroids,anti-leukemic agents, and growth factors; agents for treating diabetessuch as insulin, insulin analogues, alpha glucosidase inhibitors,biguanides, and insulin sensitizers; and agents for treatingimmunodeficiency disorders such as gamma globulin.

These additional agents may be administered separately, as part of amultiple dosage regimen, from provided chirally controlledoligonucleotides, and composition thereof. Alternatively, these agentsmay be part of a single dosage form, mixed together with the providedchirally controlled oligonucleotides, and composition thereof, in asingle composition.

The examples and preparations provided below further illustrate andexemplify provided oligonucleotides and compositions thereof, andmethods of preparing the same. It is to be understood that the scope ofthe present invention is not limited in any way by the scope of thefollowing examples and preparations.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the examples describedbelow. The following examples are intended to illustrate the benefits ofthe present invention, but do not exemplify the full scope of theinvention.

Method of Analyzing Oligonucleotide Compositions

In some embodiments, the present invention provides methods foranalyzing oligonucleotide compositions. In some embodiments, a providedmethod detects, determines, and/or quantifies, for example, theidentity, purity, quantity and/or quality of one or moreoligonucleotides. In some embodiments, the present invention provides amethod comprising steps of:

a) performing a first analysis of a first composition, which firstcomposition comprises a plurality of different types of anoligonucleotide; and

b) comparing the performed first analysis with a second analysis, undercomparable conditions as the first analysis, of a second composition,which second composition is a chirally controlled composition of theoligonucleotide, where differences between the first and second analysesreflect differences in presence or level of at least one type of theoligonucleotide in the first as compared with the second composition.

In some embodiments, a second composition contains only a single type ofthe oligonucleotide. In some embodiments, a second composition containsmore than one types of the oligonucleotide. In some embodiments, aprovided method is used for quality control of oligonucleotidecompositions, for example, as illustrated in Examples 17 and 48. As willbe appreciated by those skilled in the art, the presented data inExample 48 confirm that the depicted analyses comparing the compositionsshow that the random oligonucleotide composition (the firstcomposition), as prepared by non-chiral controlled oligonucleotidesynthesis, comprises extremely low level of certain oligonucleotidetypes, such as the full Rp or Sp type of the chirally controlledcomposition (the second composition).

EXEMPLIFICATION

The foregoing has been a description of certain non-limiting embodimentsof the invention. Accordingly, it is to be understood that theembodiments of the invention herein described are merely illustrative ofthe application of the principles of the invention. Reference herein todetails of the illustrated embodiments is not intended to limit thescope of the claims.

General Description of Oligonucleotide Synthesis

As described above and herein, in some embodiments, the presentinvention provides oligonucleotides and the methods of preparing thesame. In some embodiments, the synthesis of oligonucleotide is performedusing solid support. In some embodiments, the synthesis ofoligonucleotide is performed in solution. In some embodiments, thesynthesis of oligonucleotide comprises steps using solid support andsteps in solution phase. In some embodiments, the steps using solidsupport are performed on an oligonucleotide synthesizer.

Preparation of the Solid Support: The support (Highly Cross-linkedPolystyrene (HCP) or Controlled Pore Glass (CPG)) was used to load thefirst 5′-O-DMTr protected nucleoside contained in the oligonucleotidesequence. In some embodiments, an oxalyl group was used to link the3′-OH group of the first nucleoside to an amine group on the solidsupport, as described in Alul et al., Oxalyl-CPG: a labile support forsynthesis of sensitive oligonucleotide derivatives, Nucleic AcidsResearch 1991, 19(7), 1527. In some embodiments, the standard succinylgroup was used as the linker.

Detritylation of Nucleotide on the Solid Support: A solution of 3% TCA(trichloroacetic acid) in dichloromethane (DCM, CH₂Cl₂) was delivered tothe column containing the support which was installed on a synthesizerto de-block the 5′-O-DMTr.

CMPT-Mediated Chiral Phosphoramidite Coupling: After washing of thesolid support with anhydrous acetonitrile (ACN) and drying by reverseflush with dry argon, the free 5′-OH was coupled with the nextnucleotide in the oligonucleotide sequence, growing from the 3′ to the5′ end. This entailed co-delivery to the solid support of a solution ofthe chiral phosphoramidite together with CMPT (below), which resulted inhighly efficient coupling with excellent diastereomeric excess(typically >99%) to give a chiral phosphite diester product, asdescribed by Wada et al. J. Am. Chem. Soc., 2008, 130, 16031-1603;Angew. Chem. Intern. Ed. 2009, 48, 496-499. The solvent was normallyacetonitrile (ACN), but other solvents could also be used.

Chiral Phosphoramidites Employed in these Studies:

Additional Phosphoramidites:

Capping: In some embodiments, the capping step was carried out in twosteps. In some embodiments, the above-mentioned two capping steps werecombined into one and performed in one-pot. Unless otherwise specified,the two-step capping was used in the synthesis of the examples describedherein.

For the two-step capping, after washing of the solid support withanhydrous ACN and drying by reverse flush with dry argon, the ‘Cap A’reagent, typically comprising 5% Pac₂O solution in 2,6-lutidine/THF—1:4(v/v) was first introduced to the solid support. Without the intentionto be limited by theory, Cap A was sufficiently reactive to effectivelycap (acylate) the free amine of the chiral auxiliary. In someembodiments, such protection prevents rearrangement/decomposition of theintermediate phosphite. Immediately following this treatment, a 1:1mixture of ‘Cap A’ and ‘Cap B’ reagents was sent to the columncontaining the solid support. ‘Cap B’ was a solution of 16%N-methylimidazole (NMI) in THF which, when mixed with ‘Cap A’ effectedcapping of, for example, the unreacted 5′-OH oligonucleotide bound tothe solid support. Not wishing to be limited by theory, the capping stepwas extremely important in that, without capping of the amino group ofthe chiral auxiliary the following sulfurization step within the cycleinduced the cleavage of the internucleotidic linkage and decompositionof the oligonucleotide, for instance, due to an undesired intermolecularnucleophilic attack of the free amine of the auxiliary on thephosphorous atom. Moreover, capping of the 5′-OH groups of the run-offuncoupled solid-supported oligonucleotide shortmers is crucial inregards of obtaining high purity of the crude full-lengtholigonucleotide, avoiding low yields due to accumulation of extendedfailed sequences and tedious and difficult, if not impossible finalpurifications.

Sulfurization: After washing of the solid support with anhydrous ACN anddrying by reverse flush with dry argon, the resulting capped phosphitetriester intermediate was subject to oxidative sulfurization. A solutionof sulfurizing reagent was prepared by mixing the sulfurizing reagent,for example, an alkyl thiosulfonate derivative (300 mM), withN,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (100 mM) in ACN. Thiswas then delivered to the column containing the solid support andallowed to react for a certain amount of time (usually 5-60 min). Insome embodiments, the sulfurizing thiosulfonate reagent was used as a300 mM solution in anhydrous ACN, without addition of BSTFA. In someembodiments, the reagent solutions with and without BSTFA producedsimilar results.

Oxidation: In some embodiments, oxidation to phosphodiester wasperformed instead of the sulfurization step. Oxidation was achieved bydelivering a solution of 0.02 M I₂ in a THF/pyridine/water(70:20:10—v/v/v) co-solvent system, forming a non-chiral phosphodiesterlinkage.

5′-Deblocking of the DMTr group: DMTr removal was effected using 3% TCAin DCM. After de-blocking, the cycle can then be carried forward forfurther iterative rounds of coupling, capping, sulfurization/oxidation(either sulfurization with the same sulfurizing agent, or sulfurizationwith a different sulfurizing agent, or oxidation instead ofsulfurization, for each of the next cycles) and de-blocking.Alternatively, if the pre-designed length was reached, theoligonucleotide was subject to cycle exit and, optionally,post-synthetic treatment. In some embodiments, the oligonucleotide wasremoved before de-blocking of the terminal 5′-O-DMTr group.

Spontaneous Removal of Chiral Auxiliary: The spontaneous removal ofchiral auxiliary was achieved during sulfurization, oxidation,de-blocking of the DMTr group, or the combination thereof. No specificstep was needed to remove the chiral auxiliary.

Cleavage and Deprotection: The desired length oligonucleotide wasremoved from its solid support by cleaving the corresponding linker(oxalyl or succinyl linked HCP or CPG) whilst at the same time, theprotecting groups on the oligonucleotide were deprotected. In someembodiments, the solid-supported oligonucleotide was first treated withan anhydrous 1 M solution of 1,5-diazabicyclo(4.3.0)non-5-ene (DBN) or1,8-diazabicycloundec-7-ene (DBU) in dry ACN-trimethylsilylchloride—16:1 (v/v) for 10 min at r.t. and then washed with dry ACN, asdescribed by Reese and Yan, J. Chem. Soc., Perkin Trans. 1, 2002, 2619.In some embodiments, the material was treated with solutions of drypropylamine in dry pyridine (typically in a 1:4 ratio) for a period of18 h at r.t. or at 60° C. for 2 h. In some embodiments, either conditionafforded the crude compound with similar quality. In some embodiments,the crude oligonucleotides were cleaved from the support and deprotectedby treatment with 28% aqueous ammonia for a period of 18 h at r.t. or at60° C. for 5 h. In some embodiments, either condition afforded the crudecompound with similar quality. The solvents were then evaporated and theresidue was treated with ˜pH 1.5 aqueous solution (the pH may be alteredif desired) with 0-50% DMSO, and the crude product was subject toanalysis by a combination of HPLC and UPLC/MS. The product was purifiedby one of or a combination of reverse phase HPLC (RP-HPLC), normal phaseHPLC, ion exchange HPLC (IE-HPLC) or size exclusion chromatography. Insome embodiments, the oligonucleotide was removed from the solidsupport, deprotected and purified before de-blocking of the DMTr group.

Example 1: Synthesis of Sulfurizing Reagents and Synthesis ofPhosphoramidites

In some embodiments, the present invention provides sulfurizingreagents, and methods of making the same. In some embodiments, theprovided sulfurizing reagents were used in the synthesis ofoligonucleotides described in the present application. Exemplarysulfurizing reagents and their synthesis are illustrated in Scheme E-1

Synthesis of Compound 5

Compound 2: A solution of (Z)-but-2-ene-1,4-diol (0.93 ml, 11.3 mmol)and triethylamine (3.3 ml, 24 mmol) in dichloromethane (DCM, 50 mL) wasadded in a dropwise fashion to a stirring ice cold solution ofmethanesulfonyl chloride (1.9 ml, 24 mmol) in DCM (50 mL). Afterstirring for 0.5 h at r.t. the mixture was poured onto ice andextracted. The organic layer was collected, dried (MgSO₄) and filtered.After removal of solvent, 2.66 g compound 2 was obtained (96%), whichwas judged by NMR to be sufficiently pure for direct use in the nextstep of the reaction. ¹H NMR (399 MHz, CDCl₃) δ 5.94 (ddd, J=5.4, 4.1,1.3 Hz, 2H), 4.83 (dd, J=4.1, 1.3 Hz, 4H), 3.04 (s, 6H); ¹³C NMR 128.34,64.38, 38.27; MS (ESI +ve): calc (M+NH₄)⁺: 262.04, found: 262.05;R_(f)=0.3 (1:1 EtOAc/hexane).

Compound 3: A solution of sodium methanesulfonothioate (1.51 g, 11.3mmol) in MeOH (20 ml) was treated with neat (Z)-but-2-ene-1,4-diyldimethanesulfonate (1.25 g, 5.12 mmol) at room temperature. After 5 min,precipitation was observed to occur. After 36 h, the mixture waspartitioned between water and DCM. The organic layer was separated,dried (MgSO₄), and filtered. Removal of solvent afforded colorless oil.Column chromatography (ISCO) gave pure compound 3 (0.89 g, 63%) as acolorless oil. ¹H NMR (399 MHz, CDCl₃) δ 5.84 (ddd, J=6.6, 5.1, 1.5 Hz,2H), 3.92 (dd, J=5.1, 1.5 HZ, 4H), 3.33 (s, 6H); ¹³C NMR 128.1, 51.47,33.13; MS (ESI +ve): calc (M+NH₄)⁺: 294.00, found: 294.04; R_(f)=0.4(1:1 EtOAc/hexane).

Compound 4: Under argon atmosphere, morpholine (10 g, 115 mmol) wasadded to ethylene sulfide (15 g, 250 mmol) in a round bottom flask. Thereaction was stirred for 7 hrs and was directly loaded on to a silicagel column. The column was washed with DCM first and then 2% MeOH/DCMwas used to obtain compound 4 (15.3 g, 91%) as colorless oil. ¹H NMR(399 MHz, CDCl₃) δ 3.67-3.59 (m, 4H), 2.63-2.52 (m, 2H), 2.51-2.45 (m,2H), 2.44-2.34 (m, 4H); MS (ESI +ve): calc (M+H)⁺=148.07, found: 148.1.

Compound 5: A DCM solution (1 mL) of 2-morpholinoethanethiol (0.21 g,1.44 mmol) was added dropwise via syringe to a stirring solutioncompound 3 (0.40 g, 1.44 mmol) in DCM (10 mL) at room temperature.Immediately after addition, the reaction was checked by TLC, showingrapid formation of the product and some dimer. After 0.5 h, the mixturewas partitioned by addition of water. Upon extraction, the organic layerwas separated, dried (MgSO₄) and filtered. After removal of solvent invacuo, column chromatography gave compound 5 (0.29 g, 58%) as colorlessoil. ¹H NMR (399 MHz, CDCl₃) δ 5.78 (m, 2H), 3.92 (d, J=7.3 Hz, 2H),3.70 (t, J=4.7 Hz, 4H), 3.46 (d, J=5.5 Hz, 2H), 3.31 (s, 3H), 2.84 (dd,J=7.8, 6.7 Hz, 2H), 2.66 (dd, J=7.8, 6.7, 2H), 2.48 (t, J=4.6 Hz, 4H);¹³C NMR 130.35, 126.27, 66.97, 58.20, 53.67, 51.52, 36.22, 35.16, 33.67;MS (ESI +ve): calc (M+H)⁺: 344.05, found: 344.06; R_(f)=0.3 (EtOAc).

Compound 5b: A DCM solution (1 mL) of compound 4b (395 mg, 1.085 mmol)was added dropwise via syringe to a stirring DCM (15 mL) solutioncompound 3 (300 mg, 1.085 mmol) at r.t. After 1 h, the resultingsolution was partitioned by addition of water. Upon extraction, theorganic layer was separated, dried (MgSO₄) and filtered. After removalof the solvent in vacuo, column chromatography gave compound 5b as acolorless oil (0.35 g, 58%). ¹H NMR (399 MHz, CDCl₃) δ 5.83-5.70 (m,2H), 5.35-5.21 (dt, J=26.0, 9.3 Hz, 2H), 5.16-5.07 (m, 1H), 4.59-4.54(d, J=9.5 Hz, 1H), 4.29-4.23 (m, 1H), 4.23-4.18 (m, 1H), 3.99-3.88 (dd,J=6.7, 1.2 Hz, 2H), 3.80-3.72 (ddd, J=10.1, 4.6, 2.6 Hz, 1H), 3.64-3.56(m, 1H), 3.50-3.43 (m, 1H), 3.31 (s, 3H), 2.09 (s, 3H), 2.03 (s, 6H),2.00 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 170.68, 170.30, 169.51, 169.30,129.43, 127.14, 87.73, 76.49, 73.89, 69.16, 67.99, 61.99, 51.64, 35.89,33.58, 20.95, 20.80, 20.74, 20.71; MS (ESI +ve): calc (M+NH₄)⁺: 578.07,found: 577.96; R_(f)=0.5 (1:1 EtOAc/hexane).

Synthesis of Compound 7

Compound 6: An ice cold solution of (Z)-but-2-ene-1,4-diol (0.93 ml,11.3 mmol) and triethylamine (1.6 mL, 11.5 mmol) in DCM (50 ml) wastreated dropwise via syringe with pivaloyl chloride (1.4 ml, 11.4 mmol)over 2 min. After 1 h, TLC showed good reaction results. The resultingmixture was partitioned by addition of water. Upon extraction, theorganic layer was separated, dried (MgSO₄), and concentrated in vacuo.This crude compound was found: by TLC (Rf=0.6, 1:1 EtOAc/hexane) tocontain no starting diol and was used crude to prepare the mesylate. Thecrude material was taken up in DCM (50 ml) containing triethylamine (1.7mL, 12 mmol) and cooled on an ice bath. Methanesulfonyl chloride (0.98ml, 12.66 mmol) was added dropwise via syringe over 2 min. TLCimmediately after addition indicated complete consumption of startingmaterial. The resulting mixture was partitioned by addition of water.Upon extraction, the organic layer was separated, dried (MgSO₄),filtered and concentrated in vacuo. Column chromatography gave purecompound 6, 1.48 g, 52%, as a colorless oil. ¹H NMR (399 MHz, CDCl₃) δ5.89-5.75 (m, 2H), 4.89-4.84 (d, J=5.7 Hz, 2H), 4.68-4.63 (d, J=5.9 Hz,2H), 3.03 (s, 3H), 1.19 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 178.28,130.61, 126.11, 65.08, 59.65, 38.84, 38.21, 27.25; MS (ESI +ve): calc(M+NH₄)⁺: 268.12, found: 268.20; R_(f)=0.3 (20% EtOAc/hexane).

Compound 7: A MeOH (10 ml) solution of sodium methanesulfonothioate(0.63 g, 4.70 mmol) and (Z)-4-(methylsulfonyloxy)but-2-enyl pivalate(1.00 g, 4.00 mmol) was stirred at r.t. for 18 h with formation of awhite precipitate (after 10 min). The resulting mixture was partitionedby addition of water and DCM. Upon extraction into DCM, the organiclayer was separated, dried (MgSO4), filtered and concentrated in vacuo.Column chromatography gave compound 7, 0.83 g, 78% as a colorless oil.¹H NMR (399 MHz, CDCl₃) δ 5.82-5.73 (m, 2H), 4.73-4.66 (m, 2H),3.95-3.87 (m, 2H), 3.32 (s, 3H), 1.19 (s, 9H); ¹³C NMR (100 MHz, CDCl₃)δ 178.35, 129.37, 127.32, 59.50, 51.44, 38.84, 33.61, 27.28; MS (ESI+ve): calc (M+NH₄)⁺: 284.10, found: 284.19; R_(f)=0.4 (20%EtOAc/hexane).

Synthesis of Compound 9

Compound 9: Pivaloyl chloride (0.60 g, 5.0 mmol) was added in a dropwisefashion to a stirring solution of S-2-hydroxyethyl methanesulfonothioate(0.65 g, 4.16 mmol) in DCM (20 ml). After 2 h at r.t. the resultingmixture with white precipitate was partitioned with water. The organiclayer was separated, dried (Na₂SO₄), filtered concentrated to an oil.Column chromatography gave compound 9 as a colorless oil (0.45 g, 45%).¹H NMR (399 MHz, CDCl₃) δ 4.39-4.34 (t, J=6.3 Hz, 2H), 3.44-3.39 (t,J=6.3 Hz, 2H), 3.36 (s, 3H), 1.20 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ62.10, 51.11, 38.96, 35.19, 27.24; MS (ESI +ve): calc (M+NH₄)⁺: 158.08,found: 158.04; R_(f)=0.3 (20% EtOAc/hexane).

Synthesis of Compound 12

Compound 11: Pivaloyl chloride (4.96 ml, 40.3 mmol) was added dropwisevia syringe to an ice cold DCM solution (50 mL) of2-(hydroxymethyl)phenol (5 g, 40.3 mmol) and triethylamine (5.61 ml,40.3 mmol). An ice-cold solution of the crude pivalate ester was treatedwith triethylamine (6.74 ml, 48.4 mmol) and 50 mL DCM. Methanesulfonylchloride (3.43 ml, 44.3 mmol) was then added slowly (5 min) via syringeand the resulting mixture was warmed to r.t. The mixture was poured ontoice and the organic layer was separated then washed with sat NaHCO₃(aq),dried (MgSO₄), filtered and concentrated to afford 10.5 g crude paleyellow oil. Column chromatography (ISCO) gave pure 11 5.45 g, 47%. ¹HNMR (399 MHz, CDCl₃) δ 7.53-7.46 (dd, 7.7, 1.8 Hz, 1H), 7.46-7.40 (dt,7.7, 1.8 Hz, 1H), 7.32-7.24 (t, 7.7 Hz, 1H), 7.13-7.06 (d, 7.7 Hz, 1H),5.21 (s, 2H), 2.79 (s, 3H), 1.40 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ177.05, 150.06, 131.18, 131.07, 126.35, 125.94, 123.21, 66.88, 39.48,38.82, 27.30, 27.26. MS (ESI +ve): calc (M+NH₄)⁺: 304.12, found: 303.99;R_(f)=0.4 (20% EtOAc/hexane).

Compound 12: A MeOH (20 mL) solution of sodium methanesulfonothioate(0.825 g, 6.15 mmol) was treated with2-((methylsulfonyloxy)methyl)phenyl pivalate (1.76 g, 6.15 mmol) at r.t.and left to stir for 18 h. The mixture was partitioned between water andDCM. The organic layer was separated, dried (MgSO₄), filtered andconcentrated to afford a colorless oil. Column chromatography gave purecompound 12 as a pale colorless oil, 0.754 g, 41%. ¹H NMR (399 MHz,CDCl₃) δ 7.48-7.44 (dd, J=7.7, 1.7 Hz, 1H), 7.39-7.34 (td, J=7.8, 1.7Hz, 1H), 7.25-7.20 (td, J=7.6, 1.2 Hz, 1H), 7.10-7.06 (dd, J=8.2, 1.2Hz, 1H), 4.29 (s, 2H), 2.90 (s, 3H), 1.39 (s, 9H); ¹³C NMR (100 MHz,CDCl₃) δ 176.69, 149.59, 131.17, 129.85, 127.41, 126.18, 123.40, 51.43,39.47, 36.01, 27.30; MS (ESI +ve): calc (M+NH₄)⁺: 320.10, found: 320.09;R_(f)=0.4 (20% EtOAc/hexane).

Synthesis of Compound 14

Compound 14: Chloromethyl pivalate (0.478 ml, 3.32 mmol) was added to astirring mixture of sodium iodide (0.050 g, 0.33 mmol) and sodiummethanesulfonothioate (0.445 g, 3.32 mmol) in acetone (7 ml) at r.t.After 24 h, TLC showed good conversion to product. The solvent wasremoved, and the residue was partitioned between water and DCM. Theorganic layer was separated and dried (MgSO₄), filtered and concentratedto afford a colorless oil. Column chromatography gave pure 14 as aslightly pink solid, 0.41 g, 55%. ¹H NMR (399 MHz, CDCl₃) δ 5.67 (s,2H), 3.39 (s, 3H), 1.24 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 177.35,67.84, 52.20, 38.93, 27.05; R_(f)=0.5 (20% EtOAc/hexane).

Synthesis of Compound 16

Compound 16: Prepared from 15 and NaMTS as described previously in U.S.Pat. No. 3,484,473. ¹H NMR (399 MHz, CDCl₃) δ 4.86 (s, 2H), 3.45 (s,6H); ¹³C NMR (100 MHz, CDCl₃) δ 52.15, 41.50.

Synthesis of Compounds 18 and 19

Compound 18: Prepared from 17 and NaMTS as described previously: Chem.Pharm. Bull. Vol. 12(11) p. 1271, 1964. ¹H NMR (399 MHz, CDCl₃) δ 3.55(s, 4H), 3.40 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 50.67, 35.96.

Compound 19: A DCM solution (1 mL) of 2-morpholinoethanethiol (0.17 g,1.2 mmol) was added dropwise via syringe to a stirring solution ofcompound 18 (300 mg, 1.2 mmol) in DCM (10 mL) at r.t. Immediately afteraddition, TLC showed rapid formation of product and some dimer. After0.5 h, the mixture was partitioned by addition of NaHCO₃ solution. Uponextraction, the organic layer was separated, dried (MgSO₄), filtered andconcentrated in vacuo. Column chromatography gave pure 19 (0.20 g, 53%)as a colorless oil. ¹H NMR (399 MHz, CDCl₃) δ 3.73-3.67 (t, J=4.7 Hz,4H), 3.51-3.46 (m, 2H), 3.35 (s, 3H), 3.07-3.01 (m, 2H), 2.88-2.83 (m,2H), 2.69-2.63 (m, 2H), 2.52-2.43 (t, J=4.6 Hz, 4H); ¹³C NMR (100 MHz,CDCl₃) δ 66.96, 57.91, 53.58, 50.79, 37.66, 36.10, 35.52; MS (ESI +ve):calc (M+H)⁺: 318.03, found: 318.04; R_(f)=0.3 (EtOAc).

Synthesis of Compound 22

Compound 21: Compound 20 was converted to compound 21 by a procedureanalogous to that described for compound 11. ¹H NMR (399 MHz, CDCl₃) δ7.45-7.36 (m, 4H), 5.37 (s, 2H), 5.21 (s, 2H), 2.93 (s, 3H), 1.21 (s,9H); ¹³C NMR (100 MHz, CDCl₃) δ 178.20, 135.65, 131.92, 130.48, 129.98,129.78, 128.88, 69.05, 63.39, 38.94, 38.36, 27.27; MS (ESI +ve): calc(M+NH₄)⁺: 318.24, found: 318.14; R_(f)=0.4 (20% EtOAc/hexane).

Compound 22: Compound 21 was converted to compound 22 by a procedureanalogous to that described for compound 12. ¹H NMR (399 MHz, CDCl₃) δ7.46-7.32, (m, 4H), 5.21 (s, 2H), 4.50 (s, 2H), 3.03 (s, 3H), 1.21 (s,9H); ¹³C NMR (100 MHz, CDCl₃) δ 178.24, 135.10, 133.15, 130.93, 130.32,129.05, 129.00, 63.61, 51.07, 38.97, 38.03, 27.30; MS (ESI +ve): calc(M+NH₄)⁺: 334.11, found: 334.13; R_(f)=0.4 (20% EtOAc/hexane).

Synthesis of Compound 25

Compound 23: Compound 23 is prepared according to a literature method(Journal of Medicinal Chemistry, 50(23), 5568-5570, 2007).

Compound 24: An ice-cold pyridine solution (10 mL) of compound 23 (1mmol) is treated successively, in a dropwise fashion with acetylchloride (1 mmol), then after 5 min with MsCl (1.1 mmol). The solutionis warmed to room temperature then the solvent is removed. The residueis dissolved in EtOAc, washed with water, dried (MgSO₄), filtered andconcentrated in vacuo. Purification by column chromatography affordspure compound 24.

Compound 25: Compound 24 is converted to compound 25 by a procedureanalogous to that described for compound 12.

Synthesis of Compound 27

Compound 27: Compound 26 was converted to compound 27 by a procedureanalogous to that described for compound 14. ¹H NMR (399 MHz, CDCl₃) δ3.97 (s, 2H), 3.79 (s, 3H), 3.48 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ168.84, 53.35, 51.53, 37.83; MS (ESI +ve): calc (M+NH₄)⁺: 202.02, found:201.96; R_(f)=0.2 (20% EtOAc/hexane).

Synthesis of Compound 29

Compound 29: Compound 28 was converted to compound 29 by a procedureanalogous to that described for compound 14. ¹H NMR (399 MHz, CDCl₃) δ3.72 (s, 3H), 3.39 (t, J=6.8 Hz, 2H), 3.34 (s, 3H), 2.85 (t, J=6.8 Hz,2H); ¹³C NMR (100 MHz, CDCl₃) δ 171.53, 52.29, 50.66, 34.51, 31.20; MS(ESI +ve): calc (M+NH₄)⁺: 216.10, found: 216.04; R_(f)=0.2 (20%EtOAc/hexane).

Synthesis of Compound 31

Compound 30: Compound 30 is prepared according to a literature method(Tetrahedron, 42(2), 601-607; 1986).

Compound 31: Compound 31 is prepared from compound 30 according to apatent procedure (US 20090181444).

Synthesis of Compound 33

Compound 33: Compound 33 is prepared from compound 32 according to apatent procedure (US 20090181444).

Synthesis of Compound 38

Compound 36: An ice-cold DCM (20 mL) solution of compound 34 (1 mmol) istreated with NEt₃ (1 mmol) followed by the dropwise addition of TMS-Cl(1.1 mmol). After 1 h, the solution is washed with water, dried (MgSO₄),filtered and concentrated in vacuo. The crude TMS protected material isre-dissolved in THF (10 mL), whereon PPh₃ (1.2 mmol), compound 35 (1.2mmol), then DEAD (1.2 mmol, dropwise) are added in succession. Afterstirring at room temperature for 18 h, the solvent is removed undervacuum, the residue is re-dissolved in DCM, the solution of which iswashed with water, dried (MgSO₄), filtered and concentrated in vacuo.Purification by column chromatography affords pure compound 36.

Compound 37: A THF (10 mL) solution of compound 36 (0.5 mmol) is treatedwith TBAF (1 mmol of a 1M solution in THF), with monitoring by TLC. Oncompletion of TMS cleavage, the solvent is removed under vacuum, and theresidue is re-dissolved in DCM, the solution of which is washed withwater, dried (MgSO₄), filtered and reduced in vacuo. The crude alcoholis re-dissolved in pyridine (5 mL), and TsCl (0.55 mmol) is added. After18 h at room temperature, the solvent is removed, and the residue isre-dissolved in DCM, the solution of which is washed with water, dried(MgSO₄), filtered and reduced in vacuo. Purification by columnchromatography affords pure compound 37.

Compound 38: Compound 37 is converted to compound 38 by a procedureanalogous to that described for compound 12.

Synthesis of Compound 41

Compound 40: An ice-cold DCM (20 mL) solution of compound 39 (1 mmol) istreated with NEt₃ (1 mmol) followed by the dropwise addition of TMS-Cl(1.1 mmol). After 1 h, the solution is washed with water, dried (MgSO₄),filtered and concentrated in vacuo. The crude TMS protected material isre-dissolved in THF (10 mL), whereon PPh₃ (1.2 mmol),potassiump-toluenethiosulfonate (KTTS, 1.2 mmol), anhydrous ZnCl₂ (1mmol) then DEAD (1.2 mmol, dropwise) are added in succession. Afterstirring at r.t. for 18 h, the solvent is removed under vacuum, and theresidue is re-dissolved in DCM, the solution of which is washed withwater, dried (MgSO₄), filtered and concentrated in vacuo. Purificationby column chromatography affords pure compound 40.

Compound 41: A THF (10 mL) solution of compound 40 (0.5 mmol) is treatedwith TBAF (1 mmol of a 1M solution in THF), with monitoring by TLC. Oncompletion of TMS cleavage, the solvent is removed under vacuum, and theresidue is re-dissolved in DCM, the solution of which is washed withwater, dried (MgSO₄), filtered and concentrated in vacuo. The crudealcohol is re-dissolved in THF (10 mL), whereon PPh₃ (1.2 mmol),compound 35 (1.2 mmol), then DEAD (1.2 mmol, dropwise) are added insuccession. After stirring at r.t. for 18 h, the solvent is removedunder vacuum, and the residue is re-dissolved in DCM, the solution ofwhich is washed with water, dried (MgSO₄), filtered and concentrated invacuo. Purification by column chromatography affords pure compound 41.

Synthesis of Compound 43

Compound 43: Compound 42 is converted to compound 43 by a procedureanalogous to that described for compound 14.

Synthesis of Compound 45a, 45b, 47a and 47b

Compound 45a: A mixture of 4-(2-chloroethyl)morpholine hydrochloride(compound 44) (50 g, 269 mmol), sodium iodide (4.03 g, 26.9 mmol) andpotassium 4-methylbenzenesulfonothioate (73.0 g, 322 mmol) was stirredin MeOH (200 ml) and heated at 60° C. over 72 h. The pale yellow mixturewas cooled and diluted with 200 mL water, stirred for 0.5 h then thewhite solid was collected by filtration, washed with water (100 mL), IPA(200 mL), EtOAc (200 mL) and ether (200 mL). Dried mass=68 g. Thismicrocrystalline powder was recrystallized from 90° C. water (200 mL)and the crystalline mass was collected by filtration after standing atr.t. overnight (64 g, 71%). ¹H NMR (500 MHz, CDCl₃) δ 7.83 (d, J=8.4 Hz,2H), 7.36 (d, J=8.4 Hz, 2H), 3.67 (t, J=4.7 Hz, 4H), 3.16 (t, J=6.9 Hz,2H), 2.64 (t, J=6.9 Hz, 2H), 2.47 (s, 3H), 2.41 (t, J=4.4 Hz, 4H); ¹³CNMR (100 MHz, CDCl₃) δ 144.82, 141.99, 129.95, 127.14, 66.85, 56.54,53.18, 33.44, 21.78; MS (ESI +ve): calc (M+H)⁺: 302.08, found: 302.22.

Compound 45b: Replacing potassium 4-methylbenzenesulfonothioate withpotassium 4-chlorobenzenesulfonothioate compound 45b was synthesized bya method analogous to that described for compound 45a. (ESI +ve): calc(M+H)⁺: 324.03, 322.04, found: 324.22, 322.20 (³⁷Cl, ³⁵Cl isotopepattern).

Compound 47a: Replacing 4-(2-chloroethyl)morpholine hydrochloride with4-(2-bromooethyl)-N-methylpiperazine hydrobromide (compound 46),compound 47a was synthesized by a method analogous to that described forcompound 45a. MS (ESI +ve): calc (M+H)⁺: 315.12, found: 315.07.

Compound 47b: Replacing 4-(2-chloroethyl)morpholine hydrochloride with4-(2-bromooethyl)-N-methylpiperazine hydrobromide and potassium4-methylbenzenesulfonothioate with potassium4-chlorobenzenesulfonothioate compound 47b is synthesized by a methodanalogous to that described for compound 45a.

Synthesis of Compound 50

Compound 49: A mixture of compound 48, (500 mg, 1.894 mmol) and sodiummethanesulfonothioate (534 mg, 3.98 mmol) was dissolved in acetone (10ml) and stirred at r.t. for 4 h. TLC indicated a complete reaction. Thesolvent was removed then the mixture was partitioned by addition ofwater and DCM. Upon extraction, the organic layer was separated thendried (MgSO₄), filtered and concentrated in vacuo. Column chromatographyafforded the pure product as a colorless solid (0.60 g, 97%). ¹H NMR(399 MHz, CDCl₃) δ 7.47 (dd, J=5.5, 3.5 Hz, 2H), 7.38 (dd, J=5.5, 3.5Hz, 2H), 4.55 (s, 4H), 3.13 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 133.41,131.75, 129.51, 51.02, 37.09; MS (ESI +ve): calc (M+NH₄)⁺: 344.01,found: 344.01; R_(f)=0.5 (1:1 EtOAc/hexane).

Compound 50: A DCM solution (2 mL) compound 4, (180 mg, 1.225 mmol) wasadded dropwise via syringe to a stirring solution of compound 49 (400mg, 1.225 mmol) in DCM (20 mL) at r.t. After 0.5 h, the mixture waspartitioned by addition of NaHCO₃. Upon extraction, the organic layerwas separated then dried (MgSO₄), filtered and concentrated in vacuo.Column chromatography gave the product (170 mg, 35%) as a colorless oil.¹H NMR (399 MHz, CDCl₃) δ 7.43-7.39 (m, 1H), 7.35-7.27 (m, 3H), 4.54 (s,2H), 4.03 (s, 2H), 3.67 (t, J=4.6 Hz, 4H), 3.05 (s, 3H), 2.58-2.50 (m,4H), 2.38 (t, J=4.6 Hz, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 136.09, 133.20,131.87, 131.21, 128.86, 128.54, 66.95, 57.95, 53.52, 51.04, 40.81,38.18, 35.82; MS (ESI +ve): calc (M+H): 394.06, found: 394.23; R_(f)=0.5(EtOAc).

Synthesis of Compound 55

Compound 54: Bromine (0.246 ml, 4.78 mmol) in DCM (50 mL) was added in adropwise fashion over 0.5 h to a stirring mixture of sodium4-nitrobenzenesulfinate (2 g, 9.56 mmol) and 2-hydroxyethyl disulfide(0.585 ml, 4.78 mmol) in DCM (50 mL) at r.t. After 2 h at r.t. themixture was filtered to remove salt. The solvent was removed then columnpurification by column chromatography gave the product (2.1 g, 83%) as acolorless oil. Rf=0.4 (1:1 EA/hexane. ¹H NMR (399 MHz, CDCl₃) δ 8.41 (d,J=8.8 Hz, 2H), 8.13 (d, J=8.8 Hz, 2H), 3.89 (t, J=5.8 Hz, 2H), 3.23 (t,J=5.8 Hz, 2H), 2.02-1.94 (s, br, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 149.73,128.40, 124.82, 60.94, 39.28.

Compound 55: Compound 51 (1 g, 3.07 mmol) in DCM (50 mL) was treateddropwise with oxalyl dichloride (0.527 ml, 6.15 mmol) and stirred atr.t. During the reaction a colorless homogeneous solution was formed.After 1 h at r.t. the solvent was removed under reduced pressure. Thewhite residue (crude acid chloride, compound 52) was re-dissolved in DCM(20 mL) and added to a stirring solution of compound 54 (0.48 g, 3.1mmol) in pyridine (20 mL). After 18 h, it was judged that the reactionwas complete. The solvents were removed. The residue was re-dissolved intoluene and partitioned with water. The toluene extracts were collected,washed with brine, dried (MgSO₄), filtered and concentrated. Columnchromatography afforded pure compound 55 (0.86 g, 60%) as a colorlesssolid. ¹H NMR (399 MHz, CDCl₃) δ 8.31 (d, J=8.9 Hz, 2H), 8.03 (d, J=8.9Hz, 2H), 7.7 (d, J=7.5 Hz, 2H), 7.58 (d, J=7.5 Hz, 2H), 7.42 (t, J=7.5Hz, 2H), 7.32 (dt, J=7.5, 1.2 Hz, 2H), 5.22 (s, br, 1H), 4.34 (s, br,4H), 4.19 (t, J=6.8 Hz, 1H), 3.23 (t, J=6.0 Hz, 2H), 4.17 (s, br, 6H);¹³C NMR (100 MHz, CDCl₃) δ 173.91, 155.10, 150.60, 149.53, 143.85,141.42, 128.27, 128.20, 127.91, 127.22, 127.18, 125.12, 124.84, 120.17,66.81, 62.60, 56.40, 47.25, 34.76, 25.37; MS (ESI +ve): calc (M+H)⁺:571.11, found: 571.00; R_(f)=0.5 (EtOAc).

Synthesis of Compound 59

Compound 57: Compound 57 is prepared from compound 56 (1 mmol) byreaction with 1.5 eq Fmoc-OSu in pyridine at r.t. for 18 h. Aqueousworkup then column chromatography affords pure compound 57.

Compound 59: Compound 59 is prepared from compound 57 by a procedureanalogous to that described for compound 55.

Synthesis of Compound 63

Compound 61: Compound 61 is prepared from compound 60 by a procedureanalogous to that described for compound 57.

Compound 63: Compound 63 is prepared from compound 61 by a procedureanalogous to that described for compound 55.

Synthesis of Compound 69

Compound 65: Ethyl isobutyrate, (11.6 g, 100 mmol) was added over 0.5 hto a cooled (−78° C.) solution of LDA in THF (53 mL, 2M). The orangemixture was then warmed to 0° C. briefly before re-cooling to −78° C.1,2-dibromoethane (20 g, 106 mmol) was then added over 0.5 h to thesolution which was warmed gradually to r.t. and stirred overnight. Theresulting mixture was partitioned by addition of water and EA. Uponextraction into EA and washing with brine, the organic layer wasseparated then dried (MgSO₄), filtered and concentrated in vacuo. Columnchromatography gave the pure product as a colorless oil (6.0 g, 25%). ¹HNMR (399 MHz, CDCl₃) 4.15 (q, J=7.1 Hz, 2H), 3.35 (t, J=8.4 Hz, 2H),2.16 (t, J=8.4 Hz, 2H), 1.27 (t, J=7.1 Hz, 3H), 1.22 (s, 6H); ¹³C NMR(100 MHz, CDCl₃) δ 176.78, 60.83, 43.83, 42.89, 28.45, 25.20, 14.32;R_(f)=0.5 (5% EtOAc/hexane).

Compound 66: A mixture of compound 65 (3.4 g, 15.24 mmol) and morpholine(13.28 g, 152 mmol) in THF (20 ml) was heated at 50° C. in a glasspressure bottle over 72 h. A white precipitate was observed to form. Themixture was separated between water and EA. The EA extracts were dried(MgSO₄), filtered and concentrated. Column chromatography gave theproduct as a colorless liquid (3.5 g, 100%). ¹H NMR (399 MHz, CDCl₃)4.11 (q, J=7.1 Hz, 2H), 3.67 (t, J=4.7 Hz, 4H), 2.41 (t, br, J=4.0 Hz,4H), 2.30-2.26 (m, 2H), 1.74-1.70 (m, 2H), 1.23 (t, J=7.1 Hz, 3H), 1.17(s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 177.69, 67.11, 55.15, 54.02, 41.10,37.04, 25.49, 14.35; MS (ESI +ve): calc (M+H)⁺: 230.18, found: 230.33;R_(f)=0.5 (5% EtOAc/hexane).

Compound 67: Compound 66 (120 mg, 0.523 mmol) and sodium hydroxide (120mg, 3.00 mmol) were stirred together in 1:1 EtOH/H₂O (10 mL). After 36h, the pH of the solution was adjusted to ca. 2 by addition of c. HCl,then NEt₃ was added until the pH reached ca. 10. SiO₂ (2 g) was addedand the solvents/water were evaporated. Column chromatography with dryloading (MeOH/DCM, with 2% NEt₃ in the DCM), gave the product as apartial (approx ⅓ mol eq) HNEt₃ salt. The adjusted yield was 103 mg,84%. ¹H NMR (399 MHz, CDCl₃ plus 3 drops of DMSO-d6) δ 3.62 (t, J=4.7Hz, 4H), 2.51-2.46 (br, 4H), 2.40 (t, J=7.1 Hz, 2H), 1.62 (J=7.1 Hz,2H), 1.08 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 179.91, 65.91, 54.29,52.87, 45.44, 41.37, 35.09, 25.93, 8.56; MS (ESI −ve): calc (M−H)⁻:200.13, found: 200.16; R_(f)=0.5 (2% NEt₃/10% MeOH/DCM).

Compound 69a: A suspension of compound 67 (11 g, 54.9 mmol) in DCM (200mL) was treated with oxalyl chloride (9.42 ml, 110 mmol) and stirred atr.t. During the reaction a colorless homogeneous solution was formed.After 1 h at r.t. the solvent was removed under reduced pressure. Theyellow residue was suspended in a mixture of DCM (200 mL) and Py (100mL) then S-2-hydroxyethyl compound 54 (15.91 g, 60.4 mmol) (DB-7-5) wasadded all at once and the reaction was monitored by HPLC/MS. After 18 h,it was judged by MS and TLC that the reaction was virtually complete.The solvents were removed then the residue was redissolved inDCM/bicarbonate. The combined organics were dried (MgSO₄), filtered andreduced. Purification by column chromatography gave 12.5 g, 51% ofcompound 69a as a yellow oil which crystallized on standing. ¹H NMR (399MHz, CDCl₃) δ 8.41 (d, J=8.5 Hz, 2H), 8.13 (d, J=8.5 Hz, 2H), 4.27 (t,J=6.3 Hz, 2H), 3.65 (t, J=4.6 Hz, 4H), 3.28 (t, J=6.3 Hz, 2H), 2.40 (s,br, 4H), 2.27 (t, J=7.7 Hz, 2H), 1.71 (t, J=7.7 Hz, 2H), 1.14 (s, 6H);¹³C NMR (100 MHz, CDCl₃) δ 177.11, 150.71, 149.69, 128.34, 124.92,66.96, 61.66, 54.95, 53.92, 41.25, 46.81, 35.11, 25.36; MS (ESI +ve):calc (M+H)⁺: 447.12, found: 446.98.

Compound 69b: A suspension of compound 67 (128 mg, 0.636 mmol) in DCM(12 mL) was treated dropwise with oxalyl chloride (545 μl, 6.36 mmol)and stirred at r.t. During the reaction a colorless homogeneous solutionwas formed which soon developed a colorless precipitate. HPLC/MSindicated good conversion to acid chloride (compound 68) as shown byconversion to the N-propyl amide. The solvent was removed under reducedpressure. The white residue was cooled on an ice bath and treated with aDCM (12 mL) solution of S-2-hydroxyethyl methanesulfonothioate (compound54) (99 mg, 0.636 mmol), followed by dropwise addition of Hunig's base(0.34 g, 2.6 mmol, 4 eq) with stirring. After 1.5 h, the solution waswashed with diluted NaHCO₃(aq). The combined extracts were dried(MgSO₄), filtered and concentrated. Column chromatography gave theproduct as a colorless oil (84 mg, 39%). ¹H NMR (399 MHz, CDCl₃) δ 4.40(t, J=6.2 Hz, 2H), 3.70 (t, J=4.6 Hz, 4H), 3.44 (t, J=6.2 Hz, 2H), 3.39(s, 3H), 2.47-2.43 (br, 4H), 2.32 (t, J=7.7 Hz, 2H), 1.77 (t, J=7.7 Hz,2H), 1.64-1.58 (br, 4H), 1.22 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ177.30, 67.08, 62.27, 55.03, 54.01, 51.07, 41.32, 36.99, 35.14, 25.44;MS (ESI +ve): calc (M+H)⁺: 340.13, found: 340.27; R_(f)=0.2 (EtOAc).

Synthesis of Compound 73

Compound 70: Compound 70 was prepared by reaction of N-methyl piperazinewith compound 65 by a procedure analogous to that described for compound66. ¹H NMR (399 MHz, CDCl₃) δ 4.10 (q, J=7.1 Hz, 2H), 2.3-2.6 (br, 8H),2.28 (t, J=8.0 Hz, 2H), 2.26 (s, 3H), 1.72 (t, J=8.0 Hz, 2H), 1.23 (t,J=7.1 Hz, 3H), 1.15 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 177.71, 60.44,55.27, 54.64, 53.50, 46.18, 41.15, 37.36, 25.47, 14.34; MS (ESI +ve):calc (M+H)⁺: 243.20, found: 243.31.

Compound 71: Compound 71 was prepared by hydrolysis of compound 70 usinga procedure analogous to that described for compound 67. ¹H NMR (399MHz, CDCl₃) δ 12-10 (vbr, 1H), 3.2-2.2 (vbr, 8H), 2.44 (t, J=8.0 Hz,2H), 2.36 (s, 3H), 1.71 (t, J=8.0 Hz, 2H), 1.17 (s, 6H), ¹³C NMR (100MHz, CDCl₃) δ 181.36, 55.18, 53.51, 52.18, 44.31, 41.57, 37.19, 26.28;MS (ESI +ve): calc (M+H)⁺: 215.17, found: 215.19.

Compound 83: Bromine (2.58 ml, 50.0 mmol) in DCM (50 mL) was added in adropwise fashion over 0.5 h to a stirring mixture of sodiumbenzenesulfinate (16.4 g, 100 mmol) and 2-hydroxyethyl disulfide (6.11ml, 49.9 mmol) in DCM (50 mL) at r.t. After 2 h at r.t. TLC showed goodreaction (Rf=0.4 (1:1 EA/hexane), so the mixture was filtered to removesalt. The solvent was removed then column chromatography gave theproduct (17.4 g, 80%) as a colorless oil.

Compound 73: Compound 71 (1.342 g, 6.29 mmol), in DCM (20 mL) wastreated with oxalyl chloride (1.079 ml, 12.58 mmol) and stirred at r.t.During the reaction a pale yellow precipitate was observed to form.After 0.5 h at r.t. the solvent was removed under reduced pressure. Thepale yellow residue was suspended in a mixture of DCM (20 mL) and Py (20mL) then compound 83 (2.060 g, 9.44 mmol) was added all at once. After18 h, it was judged by TLC that the reaction was complete. The solventswere removed then the residue was redissolved in water and washed withether. The aqueous layer was reduced to a brown solid. Recrystallizationfrom boiling EtOH (10 mL) gave a slightly impure HCl salt which was freebased (NEt₃ in DCM) then loaded directly onto silica gel. Columnchromatography gave 0.95 g, 36% of pure compound 73. ¹H NMR (399 MHz,CDCl₃) δ 7.96 (d, J=8.0 Hz, 2H), 7.68 (t, J=7.2 Hz, 1H), 7.59 (t, J=7.7Hz, 2H), 4.25 (t, J=6.3 Hz, 2H), 3.25 (t, J=6.3 Hz, 2H), 2.8-2.3 (vbr,8H), 2.36 (s, 3H), 2.33 (t, J=7.7 Hz, 2H), 1.74 (t, J=7.7 Hz, 2H), 1.16(s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 177.13, 144.68, 134.09, 129.59,127.09, 61.99, 54.86, 54.35, 52.84, 45.95, 45.71, 41.26, 37.05, 34.66,25.39; MS (ESI +ve): calc (M+H)⁺: 415.17, found: 415.09.

Synthesis of Compound 76

Compound 75: Compound 74 was converted to compound 75 by a procedureanalogous to that described for compound 2. ¹H NMR (500 MHz, CDCl₃) δ6.49-6.41 (br, d, J=6.3 Hz, 1H), 4.91 (dt, J=6.3, 2.7 Hz, 1H), 4.59(ddd, J=31.4, 10.6, 3.0 Hz, 2H), 3.84 (s, 3H), 3.04 (s, 3H), 2.09 (s,3H); 170.27, 169.05, 68.90, 53.33, 52.03, 37.63, 23.16; MS (ESI +ve):calc (M+H)⁺: 240.06, found: 240.24.

Compound 76: Compound 75 was converted to compound 76 by a procedureanalogous to that described for compound 3. ¹H NMR (500 MHz, CDCl₃) δ6.56-6.40 (br, d, J=6.2 Hz, 1H), 4.93 (q, J=5.9 Hz, 1H), 3.81 (s, 3H), δ3.74 (dd, J=14.6, 4.8 Hz, 1H), 3.57 (dd, J=14.6, 5.6 Hz, 1H), 3.38 (s,3H), 2.07 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 170.44, 170.19, 53.23,52.02, 50.73, 37.77, 23.12; MS (ESI +ve): calc (M+H)⁺: 256.03, found:256.21.

Synthesis of Compound 78

A mixture of compound 77, (2.49 ml, 32.8 mmol), and sodiummethanesulfonothioate (4.4 g, 32.8 mmol) was stirred in acetone (80 ml)over 6 h. The acetone was removed by rotary evaporation and the mixturewas triturated with DCM. After filtration, DCM was removed byevaporation (5 g crude yield) and the mixture was subjected topurification by column chromatography. Yield of pure compound 78 was 2.8g, 55%, colorless oil, Rf=0.3 (20% EA/hexane). ¹H NMR (500 MHz, CDCl₃) δ5.30 (s, 2H), 3.48 (s, 3H), 3.38 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ80.07, 57.40, 22.71.

Synthesis of Compound 80

An ice-cold solution of compound 79 (5 g, 65.7 mmol) and triethylamine(11 ml, 79 mmol) in DCM (150 mL) was treated with methanesulfonylchloride (5.59 ml, 72.3 mmol) over 2 min. After 1 h the reaction wasjudged to be complete by TLC. Water was added and the organic extractswere washed sequentially with dilute HCl, saturated sodium bicarbonate,brine, then dried (MgSO₄), filtered and reduced. The product mesylate(9.4 g, 61 mmol, 96%) was taken up in acetone (200 ml), then thepotassium salt of toluene thiosulfinic acid (61 mmol) was added and thesolution was stirred at 50° C. over 18 h. A thick white precipitate wasobserved to form. The mixture was filtered, reduced to an oil thenextracted into DCM/water. The organic extracts were dried (MgSO₄),filtered and reduced. Column chromatography gave pure compound 80 as acolorless oil which crystallized on standing Rf=0.3 (20% EA/hexane)

Synthesis of Compound 82

A mixture of 2-chloro-N,N-dimethylethanamine hydrochloride (10 g, 69.4mmol), sodium iodide (1.041 g, 6.94 mmol) and potassium4-methylbenzenesulfonothioate (18.86 g, 83 mmol) was stirred in MeOH (50ml) and heated at 60° C. over 72 h. Aqueous workup then columnchromatography gave pure compound 82 (8.5 g, 47%) as a colorless oil. ¹HNMR (399 MHz, CDCl₃) δ 7.81 (d, J=8.4 Hz, 2H), 7.33 (dd, J=8.4, 0.6 Hz,2H), 3.09 (t, J=6.8 Hz, 2H), 2.53 (t, J=6.8 Hz, 2H), 2.44 (s, 3H), 2.17(s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 144.76, 142.03, 129.92, 127.18,57.51, 45.14, 34.29, 21.77; MS (ESI +ve): calc (M+H)⁺: 260.07, found:260.16.

Synthesis of Compound 85

Sodium methanesulfonate (11.5 g, 86 mmol) was placed in a dry 50 mL oneneck flask. 30 mL of anhydrous DMF (Aldrich) was added using a dry glasssyringe. 5 mL of 3-bromopropionitrile (compound 84) (8.2 g, 61.2 mmol)was added using a dry glass syringe. The reaction flask was closed underargon, sealed and stirred for 24 h at 50° C. The reaction was monitoredusing TLC (system: hexanes/ethyl acetate—5:5—v/v). The reaction mixturewas diluted with 100 mL of ethyl acetate and washed 5 times with water(5×100 mL). The organic layers were dried over sodium sulfate, filteredand evaporated to dryness. The residue was dissolved in 5 mL ofdichloromethane and purified by silica gel chromatography (CombiFlash)using a linear gradient of ethyl acetate in hexanes. Pure compound 85(7.2 g, 52%) was obtained as a colorless oil. ¹H-NMR (CDCl₃, 399 MHz). δ3.43 (s, 3H), 3.41 (t, 2H, J=2.5 Hz), 2.93 (t, 2H, J=2.5 Hz); ¹³C-NMR(CDCl₃, 100 MHz) δ 117.7, 51.2, 31.7, 19.6.

Synthesis of Compound 87

Compound 87: Compound 86 (1.11 mL, 12.5 mmol) and sodium4-nitrobenzenesulfinate (5.23 g, 25.0 mmol) were added in DCM (12.5 mL)to give a white suspension. Dibromine (0.64 ml, 12.5 mmol) was added tothe stirred solution. The solution was stirred for 30 min at r.t. andfiltrated to remove the salt, then the filtrate was evaporated underreduced pressure. The crude product was added to a silica gel column andwas eluted with hexane-EtOAc to give pure compound 87 (4.80 g, 20.6mmol, 82% yield). ¹H NMR (399 MHz, CDCl₃) δ 8.44-8.40 (m, 2H), 8.15-8.10(m, 2H), 2.58 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 149.00, 128.54,124.87, 94.61, 18.55; R_(f)=0.60 (1:1 EtOAc/hexane).

Synthesis of Compound 90

Compound 89: In a 1 L round-bottomed flask N-(2-mercaptoethyl)acetamide,88 (11.92 g, 100 mmol) was dissolved in EtOAc (300 mL) to give acolorless solution. Sodium iodide (0.150 g, 1.000 mmol) and 30% hydrogenperoxide (3.40 g, 100 mmol) in H₂O (11.3 mL) were added to the solutionwhich was stirred for 45 min at r.t. Saturated Na₂S₂O₃ was added to thesolution. The aqueous layer was extracted with EtOAc (3×300 mL). Thecombined organic layers were dried with Na₂SO₄, filtered andconcentrated by rotary evaporation. The crude product was subjected tocolumn chromatography and was eluted with a MeOH/DCM gradient to givepure compound 89 (4.94 g, 20.90 mmol, 41.8% yield), which was judged byTLC to be sufficiently pure for direct use in the next step of thereaction. R_(f)=0.35 (9:1 DCM/Methanol)

Compound 90: In a 100 mL round-bottomed flask, compound 89 (2.364 g,10.00 mmol) and sodium 4-nitrobenzenesulfinate (4.18 g, 20 mmol) weredissolved in CH₂Cl₂ (20 mL) to give a white suspension. Dibromine (0.516ml, 10.00 mmol) was added to the stirred solution. The solution wasstirred for 30 min at r.t. and filtrated to remove the salt, then thefiltrate was evaporated under reduced pressure. The crude product wasadded to a silica gel column and was eluted with hexane-EtOAc to givepure compound 90 (2.37 g, 7.79 mmol, 39% yield). ¹H NMR (399 MHz, CDCl₃)δ 8.45-8.40 (m, 2H), 8.17-8.12 (m, 2H), 3.60-3.54 (dt, 2H), 3.21-3.16(t, 2H), 2.00 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 130.35, 126.27, 66.97,58.20, 53.67, 51.52, 36.22, 35.16, 33.67; R_(f)=0.40 (EtOAc).

Synthesis of Compounds 93 and 94

Compound 92: In a 250 mL round-bottomed flask, sodium4-chlorobenzenesulfonothioate (14.5 g, 63.0 mmol) was dissolved in MeOH.Cis-1,4-dichloro-2-butene, 91 (3.16 g, 30.0 mmol) and sodium iodide (450mg, 3.0 mmol) was added to the stirred solution. The solution wasstirred for 3 h at r.t., then heated up to 50° C. After stirring for 18h at 50° C., solvents were removed under reduced pressure. The resultingcrude material was diluted with DCM (300 mL) and washed with water(1×100 mL). The aqueous layer was extracted with DCM (1×100 mL).Combined organic layers were dried with Na₂SO₄, filtered andconcentrated. The crude product was subjected to column chromatographyand was eluted with an EtOAc/hexane gradient to give compound 92 (5.99g, 12.8 mmol, 43% yield). ¹H NMR (399 MHz, CDCl₃) δ 7.87-7.84 (m, 4H),7.56-7.51 (m, 4H), 5.56-5.50 (m, 2H), 3.72-3.68 (m, 4H); ¹³C NMR (100MHz, CDCl₃) 130.35, 126.27, 66.97, 58.20, 53.67, 51.52, 36.22, 35.16,33.67; R_(f)=0.35 (1:3 EtOAc/hexane).

Compound 93: In a 100 mL round-bottomed flask compound 92 (3.09 g, 6.58mmol) was dissolved in THF at 0° C. Triethylamine (459 μL, 3.29 mmol)and 2-methyl-2-propanethiol (742 μL, 6.58 mmol) were added to thestirred solution at 0° C. The solution was stirred for 20 min at 0° C.,then triethylamine (230 μL, 1.65 mmol) was added to the stirred solutionat 0° C. After stirring for 40 min at 0° C., the solvents were removedunder reduced pressure. The resultant was subjected to columnchromatography and was eluted with an EtOAc/hexane gradient to givecompound 93 (1.77 g, 4.62 mmol, 70% yield). ¹H NMR (399 MHz, CDCl₃) δ7.90-7.85 (m, 2H), 7.56-7.52 (m, 2H), 5.73-5.65 (m, 1H), 5.55-5.48 (m,1H), 3.78-3.75 (d, 2H), 3.36-3.32 (d, 2H), 1.32 (s, 9H); ¹³C NMR (100MHz, CDCl₃) δ 143.54, 140.60, 130.90, 129.83, 128.66, 124.56, 48.29,37.05, 33.35, 30.16; R_(f)=0.60 (1:3 EtOAc/hexane).

Compound 94: Using a procedure analogous to that described for compound93, and substituting 2-propanethiol for 2-methyl-2-propanethiol, purecompound 94 was obtained (1.13 g, 3.06 mmol, 72% yield). ¹H NMR (399MHz, CDCl₃) δ 7.90-7.82 (m, 2H), 7.56-7.48 (m, 2H), 5.72-5.64 (m, 1H),5.57-5.47 (m, 1H), 3.79-3.72 (d, 2H), 3.33-3.26 (d, 2H), 3.61-2.92 (m,1H), 1.27 (d, 6H); ¹³C NMR (100 MHz, CDCl₃) 143.28, 140.36, 131.37,125.63, 124.55, 124.56, 41.32, 38.02, 35.81, 33.08, 22.54; R_(f)=0.55(1:3 EtOAc/hexane).

Synthesis of Compound 96

Compound 96: Compounds 9 (1.81 g, 5.50 mmol) and 54 (2.17 g, 8.25 mmol)were combined and dried by co-evaporation with anhydrous toluene (3×2mL). The mixture was dissolved in dry 1,2-dichloroethane (16.5 mL). 4 Åmolecular sieves were added to the solution and the mixture was stirred30 min at r.t. Trimethylsilyltriflate (497 μL, 2.75 mmol) was added tothe stirred solution. The solution was stirred for 9 h at r.t., thenadditional 54 (0.723 g, 2.75 mmol) was added to the stirred solution.After stirring 16 h at r.t., the solution was diluted with DCM (100 mL)and washed with sat. NaHCO₃ (1×100 mL). The aqueous layer was extractedwith DCM (1×50 mL). The combined organic layers were dried with Na₂SO₄,filtered and concentrated by rotary evaporation. The resulting crude wassubjected to column chromatography and was eluted with an EtOAc/hexanegradient to give compound 96 (2.21 g, 3.73 mmol, 68% yield). ¹H NMR (399MHz, CDCl₃) δ 8.42-8.36 (d, 2H), 8.12-8.06 (d, 2H), 6.07-6.00 (d, 1H),5.33-5.30 (d, 1H), 5.23-5.15 (dd, 1H), 4.69-4.63 (d, 1H), 4.15-4.04 (m,3H), 4.04-3.87 (m, 2H), 3.84-3.73 (m, 1H), 3.22-3.12 (t, 2H), 2.12-1.92(m, 12H); ¹³C NMR (100 MHz, CDCl₃) 179.95, 170.74, 170.48, 150.77,149.59, 128.47, 125.03, 101.40, 71.08, 70.02, 67.39, 66.92, 61.76,51.12, 36.51, 23.72, 20.92; R_(f)=0.25 (1:9 MeOH/DCM).

Synthesis of Compound 100

Compound 98: An ice-cold solution of compound 97 (4.87 g, 22.0 mmol) indry pyridine (70 mL) was treated with phenoxyacetic anhydride (37.8 g,132.0 mmol) and 4-dimethylaminopyridine (26.9 mg, 220 μmol). Afterstirring at r.t. for 12 h, the mixture was concentrated andco-evaporated with toluene in vacuo. The resultant was diluted by DCM(400 mL) and washed with sat. NaHCO₃ (1×400 mL). Upon extraction, theorganic layer was separated, dried with Na₂SO₄, filtered andconcentrated in vacuo. The resultant was subjected to columnchromatography and was eluted with an EtOAc/hexane gradient to give purecompound 98 (16.7 g, 22.0 mmol, 100% yield). ¹H NMR (399 MHz, CDCl₃) δ7.36-6.75 (m, 20H), 6.27-6.20 (d, 1H), 5.43-5.36 (m, 1H), 5.22-5.06 (m,2H), 4.81-4.65 (m, 3H), 4.60-4.52 (m, 2H), 4.45-4.29 (m, 2H), 4.17-4.02(m, 2H), 3.97-3.87 (m, 1H), 1.85 and 1.74 (d, 3H, rotamers); ¹³C NMR(100 MHz, CDCl₃) δ 170.20, 168.78, 168.70, 168.36, 167.52, 157.56,157.48, 157.40, 130.09, 129.70, 129.66, 129.60, 122.32, 122.01, 121.94,114.61, 114.58, 114.58, 114.39, 91.93, 68.37, 68.25, 67.30, 64.54,61.25, 46.43, 22.95; R_(f)=0.35 (1:1 EtOAc/hexane).

Compound 99: Compound 98 (15.15 g, 20.0 mmol) was dissolved inClCH₂CH₂Cl (40 mL) and trimethylsilyl trifluoromethanesulfonate (5.43mL, 30.0 mmol) was added to the stirred solution at r.t. The solutionwas stirred for 24 h at 50° C., triethylamine (12.6 mL, 90.0 mmol) wasadded and the mixture was concentrated in vacuo. The resultant waspurified by silica gel column chromatography (EtOAc (2.5% Et₃N)-hexane)to give slightly impure compound 99 containing tiny amounts ofphenoxyacetic acid. This material was used in the next step of thereaction scheme without further purification.

Compound 100: Compound 99 (3.63 g, 6 mmol) andS-2-hydroxyethyl-4-nitrobenzenesulfonothioate, 54 (2.76 g, 10.5 mmol)were dissolved in ClCH₂CH₂Cl (18 mL) to give a colorless solution. 4 ÅMolecular sieves were added to the stirred solution at r.t. The solutionwas stirred at r.t. for 30 min then trimethylsilyltrifluoromethanesulfonate (0.543 ml, 3.00 mmol) was added. The mixturewas stirred at r.t. for 24 h and TLC showed about 90% reactioncompleted. Additional 54 (237.0 mg, 0.90 mmol) was added to the mixtureand it was stirred for further 36 h to complete the reaction. Themixture was diluted with DCM (100 mL) and washed with sat NaHCO₃ (1×100mL). The aqueous layer was back-extracted with CH₂Cl₂ (1×50 mL). Thecombined organic layers were dried with Na₂SO₄, filtered, concentratedand dissolved in 120 mL of Ac₂O-pyridine (1:9, v/v). The mixture wasstirred for 12 h then concentrated under reduced pressure. The resultantwas diluted with CH₂Cl₂ (100 mL) and washed with sat NaHCO₃ (1×100 mL).The aqueous layer was back extracted with CH₂Cl₂ (1×50 mL). The combinedorganic layers were dried with Na₂SO₄, filtered and concentrated. Theresultant was purified by silica gel column chromatography (EtOAc-hexanegradient) to give pure compound 100 (2.56 g, 2.94 mmol, 49.0% yield). ¹HNMR (399 MHz, CDCl₃) δ 8.70-8.62 (d, 2H), 8.41-8.33 (d, 2H), 7.65-7.03(m, 15H), 6.35-6.27 (d, 1H), 5.81-5.72 (m, 2H), 5.06-4.97 (m, 3H),4.92-4.85 (m, 2H), 4.83-4.62 (m, 2H), 4.58-4.44 (m, 2H), 4.34-4.26 (m,2H), 4.26-4.15 (m, 1H), 4.06-3.95 (m, 1H), 3.51-3.40 (m, 2H), 2.22 and2.18 (d, 3H, rotamers); ¹³C NMR (100 MHz, CDCl₃) 171.17, 169.13, 169.05,168.92, 157.86, 157.80, 157.76, 150.82, 149.63, 130.01, 129.96, 128.47,125.06, 122.27, 122.23, 114.93, 114.74, 100.86, 70.64, 70.54, 65.36,64.90, 62.21, 51.30, 36.54, 23.74; R_(f)=0.60 (1:1 EtOAc/hexane).

Synthesis of Compound 104

Compound 102: Using a procedure analogous to that described for compound98, and substituting compound 101 for compound 97, pure compound 102 isobtained.

Compound 103 Using a procedure analogous to that described for compound99, and substituting compound 102 for compound 98, pure compound 103 isobtained.

Compound 104 Using a procedure analogous to that described for compound100, and substituting compound 103 for compound 99, pure compound 103 isobtained.

Synthesis of Compounds 109 and 111

Compound 106: A solution of compound 105 (100 mmol) in dry1,2-dichloroethane (300 mL) is treated dropwise via syringe with TiCl₄(110 mmol) over 2 min. After refluxing for 16 h, TLC shows completereaction. The crude is diluted with DCM and washed with sat. NaHCO₃. Thecombined organic layers are dried with Na₂SO₄, filtered, concentratedand used for next reaction without further purification.

Compound 107: Thiourea (150 mmol) and compound 106 (100 mmol) aredissolved in acetone (200 mL) under Ar. The reaction mixture is heatedto 60° C. and stirred. After 2 h, a white solid precipitation is removedby filtration. The precipitate is recrystallized. The filtered crystalsand Na₂S₂O₅ (140 mmol) are added to a stirred mixture of DCM (500 mL)and H₂O (250 mL). The reaction mixture is heated to reflux under Ar.After 3 h, the reaction mixture is cooled to r.t. and the phases areseparated. The aqueous layer is back extracted with DCM. The combinedorganic layers are dried over Na₂SO₄, filtered and concentrated to givecompound 107.

Compound 108: 1-Bromo-2-chloroethane (100 mmol), triethylamine (200mmol) and compound 107 (100 mmol) are dissolved in acetonitrile (200 mL)under Ar. The reaction mixture is heated to 60° C. and stirred. TLCshows completed reaction. The mixture is diluted with DCM (500 mL) andwashed with NaHCO₃ (250 mL). The organic layer is dried over Na₂SO₄,filtered, concentrated and purified to give compound 108.

Compound 109: compound 112 (120 mmol), sodium iodide (10.0 mmol) andcompound 108 (100 mmol) are dissolved in MeOH (200 mL) under Ar. Thereaction mixture is heated to 60° C. and stirred. TLC shows completedreaction. The mixture is diluted with DCM (500 mL) and washed withNaHCO₃ (250 mL). The organic layer is dried over Na₂SO₄, filtered,concentrated and purified to give compound 109.

Compound 110: 1-Bromo-4-chloro-2,3-cis-butene (100 mmol), triethylamine(200 mmol) and compound 107 (100 mmol) are dissolved in acetonitrile(200 mL) under Ar. The reaction mixture is heated to 60° C. TLC showsreaction is completed. The mixture is diluted with DCM (500 mL) andwashed with NaHCO₃ (250 mL). The organic layer is dried over Na₂SO₄,filtered, concentrated and purified to give compound 110.

Compound 111: compound 112 (120 mmol), sodium iodide (10.0 mmol) andcompound 110 (100 mmol) are dissolved in MeOH (200 mL) under Ar. Thereaction mixture is heated to 60° C. TLC shows completed reaction. Themixture is diluted with DCM (500 mL) and washed with NaHCO₃ (250 mL).The organic layer is dried over Na₂SO₄, filtered, concentrated andpurified to give compound 111.

Synthesis of Compound 115

Compound 113: 2-Chlorosulfonylacetonitrile (113) is prepared followingthe procedure of Sammes (as described in patent GB 1252903).

Compound 114: Sodium sulfide nonahydrate (65 mmol) is taken up in 100 mLwater. The flask is kept in a water bath. Compound 113 (50 mmol) isadded dropwise to the solution while stirring and gently warming thewater bath. Sulfur is observed to appear and then disappear in theflask. Solvent is evaporated under vacuum and the residue isrecrystallized from ethanol. Sodium cyanomethanesulfonothioate (compound114) is thus obtained as a colorless crystalline solid.

Compound 115: Compound 114 (13.69 g, 86 mmol) is taken up, withstirring, in anhydrous DMF (30 mL) in a 100 mL round bottom flask. Then,3-bromopropionitrile (5 mL, 8.2 g, 61.2 mmol) is added and the resultingmixture is stirred for 18 h at 50° C. with monitoring by TLC. Oncompletion of reaction (complete consumption of 3-bromopropionitrile),the reaction mixture is diluted with EtOAc (100 mL) and the organiclayer is washed with 5×20 mL H₂O. The organic separated organic layer isthen dried (MgSO₄), filtered and reduced to an oil by rotaryevaporation. The crude oil is purified by column chromatography using anEtOAc/hexane gradient and the fractions containing pure material arecollected and the solvent is removed by rotary evaporation then furtherdrying in vacuo to furnish the pure compound 115 as a colorless oil.

Synthesis of Compound 118

Compound 116: Compound 51 (2.9 g, 8.91 mmol) in DCM (50 mL) was treateddropwise with oxalyl dichloride (0.53 ml, 6.15 mmol) then DMF (10 μL)and stirred at r.t. During the reaction a colorless homogeneous solutionwas formed. After 2 h at r.t. the solvent was removed under reducedpressure. The white residue was redissolved in DCM (20 mL) then addeddropwise to a stirring pyridine (10 mL) solution of ethane-1,2-dithiol(8.40 g, 89 mmol), with monitoring by UPLC/MS. After 18 h, it was judgedthat the reaction was complete. The solvents were removed and theremaining ethanethiol was removed by trap-to-trap distillation then theresidue was subjected to purification by column chromatography with DCMto provide the pure compound 116 as a colorless solid. Yield was 1.82 g,51%. MS (ESI+): calc (M+Na)⁺: 424.10, found: 424.06. ¹H NMR (500 MHz,CDCl₃) δ 7.79 (d, J=7.5 Hz, 2H), 7.63 (d, J=5.7 Hz, 2H), 7.43 (t, J=7.5Hz, 2H), 7.34 (t, J=7.4 Hz, 2H), 5.40-5.15 (br, 1H), 4.60-4.35 (br, 2H),4.31-4.19 (m, 1H), 3.15-3.04 (br, 2H), 2.95-2.80 (m, 1H), 2.75-2.60 (br,2H), 1.72-1.43 (m, 6H); ¹³C NMR (126 MHz, CDCl₃) 203.14, 154.75, 143.92,141.49, 127.84, 127.19, 125.17, 120.13, 66.81, 62.69, 47.41, 33.01,25.78, 24.60.

Compound 117: To a stirred solution of compound 116 (1.7 g, 4.23 mmol)in EtOAc (12 mL) was added sodium iodide (6.35 mg, 0.042 mmol) andhydrogen peroxide (0.144 g, 4.23 mmol) (0.45 mL of a 30% aqueoussolution) and the mixture was stirred at r.t. for 15 min. TLC showedcomplete consumption of the starting material. Aqueous sodiumthiosulfate was added until the solution became colorless. The solutionwas washed with water, dried (MgSO₄), then column chromatography(EtOAc/hexane), gave pure compound 117 as a colorless solid foam (1.45g, 86%). MS (ESI+): calc (M+H)⁺: 802.07, found: 802.08. ¹H NMR (500 MHz,CDCl₃) δ 7.78 (d, J=7.6 Hz, 4H), 7.63 (d, J=5.8 Hz, 4H), 7.42 (t, J=7.5Hz, 4H), 7.33 (t, J=7.5 Hz, 4H), 5.42-5.25 (br, 2H), 4.55-4.35 (br, 4H),4.30-4.18 (br, 2H), 3.25-3.10 (br, 4H), 2.95-2.70 (br, 4H), 1.65-1.45(br, 12H); ¹³C NMR (126 MHz, CDCl₃) δ 203.19, 154.71, 143.90, 141.45,127.80, 127.17, 125.16, 120.10, 66.76, 62.65, 47.37, 37.90, 28.56,25.73.

Compound 118: Dibromine (0.091 ml, 1.77 mmol) was added in a dropwisefashion over 2 min to a stirring mixture of sodium4-nitrobenzenesulfinate (0.742 g, 3.55 mmol) and compound 117 (1.42 g,1.773 mmol) in DCM (10 mL) at r.t. After 30 min stirring at r.t., themixture was filtered and the filtrate was reduced to an orange solidfoam in vacuo. Column chromatography (DCM/hexane) gave pure compound 118(1.26 g, 60%) as a pale yellow solid foam. MS (ESI+): calc (M+H)⁺:587.71, found: 802.08. ¹H NMR (500 MHz, CDCl₃) δ 8.36 (d, J=8.2 Hz, 2H),8.14 (d, J=7.3 Hz, 2H), 7.79 (d, J=7.0 Hz, 2H), 7.63 (s, 2H), 7.43 (t,J=6.7 Hz, 2H), 7.35 (d, J=6.8 Hz, 2H), 5.35-5.15 (br, 1H), 4.55-4.35(br, 2H), 4.30-4.20 (br, 1H), 3.30-3.00 (br, 4H), 1.70-1.25 (br, 6H);¹³C NMR (126 MHz, CDCl₃) δ 202.71, 154.66, 150.60, 149.63, 143.78,141.46, 128.40, 127.91, 127.21, 125.12, 124.81, 120.18, 66.86, 62.59,47.35, 35.84, 28.42, 25.61.

Synthesis of Compound 120

Compound 120: Commercially available compound 119 (1 g, 7.04 mmol) andpotassium 4-methylbenzenesulfonothioate (1.753 g, 7.75 mmol) werestirred in MeOH (10 mL) over 5 days at r.t. then at 40° C. over 24 h.TLC showed good conversion to product. The MeOH was evaporated and theresidue was taken up in DCM (30 mL) and Boc₂O (1.54 g, 7.04 mmol) wasadded. The mixture was treated with triethylamine (1.030 ml, 7.39 mmol)in a dropwise fashion over 10 min at r.t. until the solution becamepractically homogeneous. Washing with water (50 mL), drying (MgSO₄),filtration and evaporation gave the crude product which was furtherpurified by column chromatography to give pure compound 120 (1.64 g,65%) as a colorless solid. MS (ESI+): calc (M+Na)⁺: 380.10, found:380.11. ¹H NMR (399 MHz, CDCl₃) δ 7.80 (d, J=8.2 Hz, 2H), 7.34 (dd,J=8.5, 0.8 Hz, 2H), 5.54 (ddt, J=24.7, 16.6, 7.9 Hz, 2H), 4.65 (s, 1H),3.77-3.66 (m, 4H), 2.45 (s, 3H), 1.43 (s, 9H); ¹³C NMR (100 MHz, CDCl₃)δ 155.83, 145.05, 142.06, 131.80, 130.01, 127.15, 124.55, 79.72, 37.29,32.75, 28.51, 21.79.

Synthesis of Compound 122

Compound 122: A solution of commercially available compound 121 (1 g,3.59 mmol) in water (1 mL) was treated all at once with a solution ofpotassium hexafluorophosphate (0.662 g, 3.59 mmol) in water (10 mL) andagitated by shaking at r.t. for 5 min. The resulting solid was collectedby filtration, washed with 2×3 mL water and dried in vacuo over KOH togive pure compound 122 as a colorless solid (1.01 g, 82%). ¹H NMR (300MHz, CD₃CN) δ 3.67-3.60 (m, 2H), 3.49 (s, 3H), 3.53-3.45 (m, 2H), 3.10(s, 9H); ¹³C NMR (126 MHz, CD₃CN) δ 65.98, 54.05 (q, J=4.1 Hz), 51.11,28.99; ¹⁹F NMR (376 MHz, CD₃CN) δ −73.15 (d, J=706.5 Hz); ³¹P NMR (162MHz, CD₃CN) δ −143.50 (hept, J=706.6 Hz).

Synthesis of Compound 125

Compound 124: A DMF (50 mL) solution of commercially available compound123 (10 mmol) is treated successively with tert-butyl-2-aminoacetate (22mmol), diisopropylethylamine (50 mmol) and HBTU (24 mmol). After 1 hstirring at r.t., water (100 mL) is added and the resulting solution isextracted with EtOAc (2×50 mL) and washed with 5% NaHCO₃ (2×25 mL) thendilute brine (2×25 mL), dried (MgSO₄), filtered and reduced by rotaryevaporation. Purification by column chromatography provides the purecompound 124.

Compound 125: Dibromine (0.091 ml, 1.77 mmol) is added in a dropwisefashion over 2 min to a stirring mixture of sodium4-nitrobenzenesulfinate (0.742 g, 3.55 mmol) anddi-tert-butyl-2,2′-((4,4′-disulfanediyl-bis(butanoyl))bis(azanediyl))diacetate(824 mg, 1.77 mmol) in DCM (10 mL) at r.t. After 30 min stirring atr.t., the mixture is filtered and the filtrate is reduced to a solidfoam in vacuo. Column chromatography gives the pure product as a solidfoam.

Synthesis of Compound 129

Compound 127: A solution of ethane-1,2-dithiol (1.130 g, 12 mmol) in TFA(10 mL) was treated all at once with commercially available compound 126(0.89 g, 9.99 mmol). The solution became warm. After 1 h stirring atr.t. the volatiles were removed and the residue was subjected to columnchromatography to give pure compound 127 as a colorless oil (0.95 g,48%). R_(f)=0.6 (5% MeOH/DCM); ¹H NMR (500 MHz, CDCl₃) δ 6.09-5.93 (br,1H), 4.41 (d, J=6.4 Hz, 2H), 2.87-2.83 (m, 2H), 2.79 (ddd, J=9.7, 7.3,3.5 Hz, 2H), 2.04 (s, 3H), 1.71 (t, J=8.0 Hz, 1H); ¹³C NMR (126 MHz,CDCl₃) δ 170.25, 40.89, 35.22, 24.91, 23.44.

Compound 129: A warm (35° C.) DMF (10 mL) solution of commerciallyavailable compound 128 (939 mg, 3.0 mmol) was treated in a dropwisefashion over 5 min with a DCM (5 mL) solution of compound 127 (250 mg,1.513 mmol). After checking TLC, the solvents were removed by rotaryevaporation under high vacuum then the residue was triturated with DCM(50 mL), filtered, and the filtrate was washed with 5×5% NaHCO₃, thenwas dried (MgSO₄), filtered and reduced to a pale yellow solid. Columnchromatography gave pure compound 129 as a pale yellow solid. MS (ESI+):calc (M+H)⁺: 320.02, found: 320.34; ¹H NMR (500 MHz, CDCl₃) δ 9.28 (dd,J=2.7, 0.7 Hz, 1H), 8.43 (dd, J=8.9, 2.6 Hz, 1H), 7.93 (dd, J=8.9, 0.7Hz, 1H), 6.10-5.90 (br, 1H), 4.41 (d, J=6.5 Hz, 2H), 3.13 (dd, J=8.6,6.3 Hz, 2H), 2.93 (dd, J=8.6, 6.4 Hz, 2H), 2.00 (s, 3H); ¹³C NMR (126MHz, CDCl₃) δ 170.18, 168.57, 145.22, 142.22, 131.81, 119.59, 40.65,38.59, 30.07, 23.39.

Synthesis of Compound 134

Compound 130: Compound 130 is prepared by a previously described method(Heterocycles, Vol 54, p 139, 2001).

Compound 132: Using an analogous procedure described previously for thesynthesis of compound 126 (Organic Syntheses, Coll. Vol. 6, p. 5(1988)), compound 131 (Tetrahedron Letters, 48(39), 7038-7041; 2007) isconverted to compound 132 by gently heating in the presence of anaqueous formaldehyde solution containing K₂CO₃.

Compound 133: Using a procedure analogous to that described for thesynthesis of compound 127 and substituting compound 132 for compound 126and compound 130 for ethane-1,2-dithiol, pure compound 133 is thusobtained.

Compound 134: Using a procedure analogous to that described for thesynthesis of compound 129 and substituting compound 133 for compound127, pure compound 134 is thus obtained.

Synthesis of Compound 140

Compound 136: An ice-cold solution of commercially available compound135 (50 g, 342 mmol), and methanol (277 mL) was treated dropwise withsulfuric acid (3.36 g, 34.2 mmol) over 10 min then warmed to r.t.gradually overnight. Most of the solvent was removed by rotaryevaporation, then saturated aqueous NaHCO₃ was carefully added. The pHof the solution was adjusted to 1.9 by addition of 1 M HCl thenextracted into EtOAc (200 mL), washed with diluted brine (50 mL), dried(MgSO₄), filtered and reduced to 29 g of colorless oil. Columnchromatography gave pure compound 136 as a colorless solid (12.5 g,23%). ¹H NMR (500 MHz, CDCl₃) δ 12.5-10.5 (vbr, 1H), 3.69 (s, 3H), 2.63(s, 2H), 1.32 (s, 6H); ¹³C NMR (126 MHz, CDCl₃) δ 183.32, 171.76, 51.72,43.88, 40.60, 25.34.

Synthesis of Compound 138: Compound 136 (10 mmol) in DCM (50 mL) istreated dropwise with oxalyl dichloride (10 mmol) then DMF (10 μL) andstirred at r.t. During the reaction a colorless homogeneous solution isformed. After 2 h at r.t. the solvent is removed under reduced pressure.The white residue is redissolved in DCM (20 mL) then added dropwise to astirring pyridine (10 mL) solution of compound 137 (10 mmol), withmonitoring by TLC. After 18 h, the solvents are removed and the residueis subjected to purification by column chromatography with DCM toprovide the pure compound 137.

Compound 139: Compound 138 (5 mmol) as a solution in THF (20 mL) isadded to an ice-cold solution of LiBH₄ (5 mmol) in THF (20 mL). Progressof the reaction at r.t. is judged by TLC. After completion of thereaction (complete consumption of starting material), water (100 mL) isadded carefully to the ice-cold solution which is subsequently extractedwith EtOAc (2×100 mL). The combined organic extracts are dried (MgSO₄),filtered and the solvents are removed by rotary evaporation. The residueis subjected to column chromatography the pure compound 139 is thusobtained.

Compound 140: Using a procedure analogous to that described for thesynthesis of compound 80, and substituting compound 139 for compound 79,pure compound 140 is thus obtained.

Synthesis of Compound 143

Compound 139: Using a procedure analogous to that described for thesynthesis of compound 139 and substituting compound 136 for compound138, pure compound 141 is thus obtained.

Compound 142: A solution of compound 141 (8.39 mmol) in DCM/MeOH (12/3mL) is treated dropwise at 0° C. with a 2 M solution of(diazomethyl)trimethylsilane (1.05 g, 9.23 mmol) in ether over 30 min.After quenching excess reagent with AcOH, washing with water (2×5 mL),drying (MgSO₄), filtration, removal of solvent by rotary evaporation,then column chromatography the pure compound 142 is obtained.

Compound 143: Using a procedure analogous to that described for thesynthesis of compound 80, and substituting compound 142 for compound 79,pure compound 143 is thus obtained.

Synthesis of Compound 147

Compound 144: Using a procedure analogous to that described for thesynthesis of compound 69a, and substituting compound 136 for compound67, and compound 54 for NH₄Cl plus iPr₂NEt, pure compound 144 is thusobtained.

Compound 145: LiAlH₄ (100 mL of a 2 M solution in THF) is added in adropwise fashion to an ice-cold THF (500 mL) solution of compound 144(86 mmol). The mixture is warmed to r.t. then refluxed for 0.5 h. Theresidual LiAlH₄ is destroyed by careful addition of saturated Na₂SO₄ tothe ice-cold solution until a granular precipitate is formed. Themixture is filtered and reduced then redissolved in EtOAc, dried(MgSO4), filtered and reduced in vacuo to furnish compound 145 which issufficiently pure for direct use in the next step of the reaction.

Compound 146: Using a procedure analogous to that described for thesynthesis of compound 124, and substituting compound 145 for compound123, and Leu-Fmoc-OH for tert-butyl 2-aminoacetate, pure compound 146 isthus obtained.

Compound 147: Using a procedure analogous to that described for thesynthesis of compound 80, and substituting compound 146 for compound 79,and compound 114 for potassium 4-methylbenzenesulfonothioate, purecompound 147 is thus obtained.

Synthesis of Compound 150

Compound 148: Commercially available chloromethyl pivalate (10 mmol) isadded in a dropwise fashion to a stirring solution of compound 130 (20mmol) in 1,2-dichloroethane (100 mL) under Ar. The resulting solution isheated in order to force to completion (noted by disappearance ofchloromethylpivalate material by TLC). Solvent is removed by rotaryevaporation, then the residue is subjected to column chromatography toprovide the pure compound 148.

Compound 149: Using a procedure analogous to that described for thesynthesis of compound 89, and substituting compound 148 for compound 88,pure compound 149 is thus obtained.

Compound 150: Using a procedure analogous to that described for thesynthesis of compound 87, and substituting compound 149 for compound 86,pure compound 150 is thus obtained.

Additional Sulfurization Reagents:

wherein S^(a) denotes any of the following structures:

Synthesis of Phosphoramidites

In some embodiments, the present invention provides phosphoramidites,and methods of making the same. In some embodiments, the providedphosphoramidites were used in the synthesis of oligonucleotidesdescribed in the present application. Exemplary phosphoramidites andtheir synthesis are described below.

Synthesis of Compound 203

Compound 202: N⁴-Bz-5′-O-DMTr-2′-O-MOE-5-methylcytidine (201) (2.00 g,2.77 mmol) was treated with 2 M ammonia in 2-propanol (Aldrich,anhydrous, 45 mL) and Pyridine (22.5 mL) at 60° C. for 5 h then at r.t.overnight, with monitoring by TLC and UPLC/MS. Solvent was removed (3×azeotrope with toluene) then column chromatography (0-100/MeOH/DCM) gavethe pure compound 202 (1.62 g, 95%) as a colorless solid which washomogeneous by TLC and HPLC. (ESI+): calc (M+H)⁺: 618.28, found: 618.53.¹H NMR (399 MHz, CDCl₃) δ 7.82 (d, J⁼1.3 Hz, 1H), 7.47-7.40 (m, 2H),7.37-7.18 (m, 8H), 6.83 (dd, J=8.9, 1.7 Hz, 4H), 5.93 (d, J=1.1 Hz, 1H),4.43 (td, J=8.3, 4.9 Hz, 1H), 4.24 (ddd, J=11.7, 5.1, 3.0 Hz, 1H), 4.07(dt, J=8.6, 2.4 Hz, 1H), 4.03-3.98 (m, 1H), 3.90 (ddd, J=11.7, 6.3, 3.3Hz, 1H), 3.79 (d, J=1.0 Hz, 6H), 3.63-3.52 (m, 3H), 3.44 (dd, J=11.0,3.0 Hz, 1H), 3.37 (s, 3H), 3.32 (d, J=9.4 Hz, 1H), 1.80-1.64 (s, br,1H), 1.13 (s, 3H).

Compound 203: Isobutyric anhydride (0.48 ml, 2.9 mmol) was addeddropwise to a solution of compound 202 (1.61 g, 2.61 mmol) in DMF (13ml) at r.t. The solution was allowed to stir at r.t. over 24 h then thesolvent was removed in vacuo at 35° C. Extraction into ether/bicarbonatethen drying (MgSO₄), filtration and removal of the solvent gave thecrude product as a colorless solid foam. Column chromatography (0-4%MeOH/DCM) gave the pure product (1.75 g, 98%) as a colorless solid foamwhich was homogenous by TLC and HPLC. (ESI+): calc (M+H)⁺: 688.32,found: 688.56. ¹H NMR (399 MHz, CDCl₃) δ 8.00-7.75 (br, 1H), 7.45-7.39(m, 2H), 7.34-7.20 (m, 8H), 6.84 (dd, J=9.0, 1.2 Hz, 4H), 5.97 (s, 1H),4.43 (d, J=6.1 Hz, 1H), 4.20-4.04 (m, 3H), 3.79 (d, J=0.9 Hz, 6H),3.64-3.58 (m, 1H), 3.64-3.52 (m, 2H), 3.47-3.40 (m, 2H), 3.38 (s, 3H),1.70-1.55 (br, 1H), 1.39 (d, J=1.0 Hz, 3H), 1.18 (d, J=6.9 Hz, 6H).

Synthesis of Compound 205

Compound 205: (R)-1-phenyl-1-((S)-pyrrolidin-2-yl)ethanol (4) was driedby azeotropic distillation with toluene (3×3 mL). A solution driedcompound 4 (0.725 g, 3.79 mmol) and 4-methylmorpholine (0.833 ml, 7.58mmol) in toluene (5 mL) was added to an ice-cold solution oftrichlorophosphine (0.331 ml, 3.79 mmol) in toluene (5 mL) which wasthen warmed to r.t. for 1 h then filtered under Ar and reduced to an oilwhich was used in the next step of the reaction without furtherpurification.

Synthesis of Compound 207

Compound 207: Using a procedure analogous to that described for thesynthesis of compound 205, and substituting compound 206 for compound204, compound 207 was obtained as a crude brown oil and was used in thenext step of the reaction without further purification.

Synthesis of Compound 208

Compound 208: Compound 203 (1.74 g, 2.53 mmol) was dried byco-evaporation with pyridine (3×5 mL) then toluene (5×5 mL). Theresulting dried 203 was dissolved in THF (15 mL), then triethylamine(2.47 ml, 17.7 mmol) was added and the solution was cooled to −78° C. bymeans of CO₂(s)/acetone cooling bath. A THF solution (15 mL) of thecrude compound 205 (3.79 mmol) was added dropwise over 0.5 h thengradually warmed to r.t. After 1 h at r.t., TLC indicated completeconversion of compound 203 to product. The mixture was washed into aseparation funnel with chloroform (100 mL) then was extracted withNaHCO₃ (saturated, aqueous, 50 mL then 2×25 mL). At each extraction, theaqueous extracts were washed with an additional 1×10 mL of chloroform.The combined chloroform extracts were dried (MgSO₄), filtered, andconcentrated by rotary evaporation at 32° C. The crude solid thusobtained was redissolved in DCM containing a few drops of triethylaminethen subjected to column chromatography with a hexane/EtOAc gradientcontaining a steady concentration of 2% triethylamine to give purecompound 208 as a white solid foam. ¹H NMR (399 MHz, CD₃CN) δ 7.92-7.80(s, 1H), 7.52-7.47 (m, 2H), 7.40-7.24 (m, 12H), 6.88 (dd, J=9.0, 1.1 Hz,4H), 5.94 (d, J=3.9 Hz, 1H), 4.79 (dt, J=9.6, 5.6 Hz, 1H), 4.31-4.27 (m,1H), 4.27-4.22 (m, 1H), 3.87 (t, J=4.7 Hz, 2H), 3.75 (d, J=2.0 Hz, 6H),3.66 (td, J=5.9, 5.5, 2.2 Hz, 1H), 3.58-3.53 (m, 2H), 3.50 (dd, J=11.1,2.4 Hz, 1H), 3.38 (dd, J=11.0, 3.3 Hz, 1H), 3.31 (s, 3H), 2.85 (dq,J=10.5, 7.4, 6.9 Hz, 2H), 1.73 (s, 3H), 1.52 (d, J=1.0 Hz, 3H),1.47-1.32 (m, 2H), 1.17 (dd, J=6.9, 0.7 Hz, 6H), 1.05-0.90 (m, 2H); ³¹PNMR (162 MHz, CD₃CN) δ 155.35.

Synthesis of Compound 210

Compound 210: Using a procedure analogous to that described for compound208 and substituting compound 209 for compound 203 and compound 207 forcompound 205, pure compound 210 was obtained as a colorless solid foam.¹H NMR (399 MHz, CD₃CN) δ 8.31-8.27 (m, 2H), 7.86 (d, J=1.2 Hz, 1H),7.62-7.55 (m, 1H), 7.55-7.45 (m, 5H), 7.44-7.24 (m, 12H), 6.94-6.90 (m,4H), 5.96 (d, J=3.7 Hz, 1H), 4.86-4.76 (m, 1H), 4.29 (dd, J=5.0, 3.8 Hz,1H), 4.23 (dt, J=5.8, 2.8 Hz, 1H), 3.92-3.80 (m, 2H), 3.78 (s, 6H), 3.74(ddd, J=7.1, 5.3, 2.1 Hz, 1H), 3.55 (t, J=4.7 Hz, 2H), 3.51 (d, J=2.3Hz, 1H), 3.41 (dd, J=11.1, 3.4 Hz, 1H), 3.30 (s, 3H), 3.26 (ddd, J=10.3,6.1, 2.2 Hz, 1H), 2.86 (ddt, J=10.1, 8.2, 7.2 Hz, 1H), 1.72 (d, J=0.7Hz, 3H), 1.62 (d, J=1.1 Hz, 3H), 1.56-1.41 (m, 2H), 1.09-0.87 (m, 2H);³¹P NMR (162 MHz, CD₃CN) δ 155.22.

Synthesis of Compound 212

Compound 212: Using a procedure analogous to that described for compound208 and substituting compound 211 for compound 203, pure compound 212was obtained as a colorless solid foam. ¹H NMR (399 MHz, CDCl₃) δ9.25-8.70 (br, 1H), 7.69 (d, J=1.3 Hz, 1H), 7.46-7.40 (m, 2H), 7.40-7.17(m, 12H), 6.81 (dd, J=9.0, 2.5 Hz, 4H), 6.09 (d, J=4.3 Hz, 1H), 4.78(dt, J=9.6, 5.2 Hz, 1H), 4.36-4.26 (m, 2H), 3.94-3.88 (m, 2H), 3.74 (dd,J=4.0, 0.9 Hz, 6H), 3.69 (td, J=6.4, 2.9 Hz, 1H), 3.64-3.56 (m, 3H),3.44-3.37 (m, 2H), 3.36 (d, J=0.9 Hz, 3H), 3.00 (dtd, J=10.3, 7.9, 6.4Hz, 1H), 1.72 (s, 3H), 1.54-1.44 (m, 1H), 1.38 (dd, J=6.8, 5.4 Hz, 1H),1.34 (d, J=1.2 Hz, 3H), 1.23-1.13 (m, 1H), 0.97-0.87 (m, 1H); ³¹P NMR(162 MHz, CDCl₃) δ 158.18.

Synthesis of Compound 213

Compound 213: Using a procedure analogous to that described for compound210 and substituting compound 211 for compound 209, pure compound 213was obtained as a colorless solid foam. ¹H NMR (399 MHz, CDCl₃) δ9.55-9.10 (br, 1H), 8.09 (d, J=1.5 Hz, 1H), 7.80-7.71 (m, 2H), 7.69-7.47(m, 12H), 7.20-7.08 (m, 4H), 6.32 (d, J=2.9 Hz, 1H), 5.20-5.11 (m, 1H),4.66-4.59 (m, 1H), 4.49-4.39 (m, 1H), 4.33-4.24 (m, 1H), 4.16-4.02 (m,8H), 4.00-3.93 (m, 1H), 3.91-3.84 (m, 2H), 3.73 (dd, J=11.0, 2.5 Hz,1H), 3.65-3.52 (m, 4H), 3.27-3.16 (m, 1H), 2.15 (s, 3H), 1.88-1.76 (m,1H), 1.76-1.65 (m, 1H), 1.63-1.49 (m, 4H), 1.29-1.18 (m, 1H); ³¹P NMR(162 MHz, CDCl₃) δ 160.08.

Synthesis of Compound 215

(i) TMSCl, NEt3, 0° C.; (ii) 2,4,6-trimethylbenzene-1-sulfonyl chloride,DMAP; (iii) 1-methylpyrrolidine, 3-hydroxypropanenitrile, DBU, 0° C.;(iv) MeOH/H2O, 4 d, room temperature

Compound 215: (i) Transient TMS Protection: A solution ofN²-isobutyryl-5′-O-DMTr-2′-O-MOE-guanosine (compound 214) (15.04 g, 21.1mmol) and triethylamine (11.8 ml, 85 mmol) was taken up in ACN (200 mL)then chlorotrimethylsilane (10.4 ml, 82 mmol) was added dropwise to theice-cold solution which was stirred at r.t. for 1 h with monitoring byTLC. Filtration and removal of solvent, then extraction (DCM/NaHCO₃500mL/200 mL), drying (MgSO₄), filtration, and removal of the solvent gavethe crude compound which was used in the next step of the reactionwithout further purification. The compound was homogeneous by TLC(R_(f)=0.6, (5% MeOH/DCM)). (ii) Activation of O⁶ position forCyanoethyl protection: The residue was redissolved in DCM (500 mL) thentriethylamine (13.1 ml, 94 mmol) was added, followed by2,4,6-trimethylbenzene-1-sulfonyl chloride (6.15 g, 28.1 mmol) and DMAP(0.14 g, 1.146 mmol). The mixture was stirred at r.t. for 3 h withmonitoring by TLC until full disappearance of the starting material andemergence of a new spot by TLC (R_(f)=0.9, (5% MeOH/DCM)). (iii)Cyanoethyl protection: 1-methylpyrrolidine (22.43 ml, 211 mmol) wasadded slowly in a dropwise fashion to the ice-cold solution with furtherreaction at 0° C. over 1 h, monitoring by TLC (new spot, R_(f)=0.2,streak, (5% MeOH/DCM)). 3-hydroxypropanenitrile (14.40 ml, 211.0 mmol)then DBU (6.32 mL, 42.1 mmol) were added in a dropwise fashionsuccessively to the still ice-cold solution and stirring was continuedfor additional 90 min, monitoring by TLC (new spot, R_(f)=0.6, (5%MeOH/DCM)). In order to force the reaction almost to completion, afurther 1.6 mL of DBU was added dropwise with careful monitoring by TLC.The mixture was poured onto NaH₂PO₄ (50 g in 400 mL water), the organicswere separated, and washed with dilute NaH₂PO₄ (10 g in 200 mL water),dried (MgSO₄), filtered and reduced by rotary evaporation. (iv) TMSDeprotection: The residue thus obtained in the previous step wasredissolved in MeOH (250 mL) then water was added in a dropwise fashionbeing careful not to induce precipitation. The dropwise addition wascontinued over 4 days with monitoring by TLC. Most of the solvent wasremoved and the residue was extracted (DCM/NaHCO₃ (500/200 mL), filteredand reduced. The residue was subjected to column purification (0-5%MeOH/DCM) to give pure compound 215 as a white foam which washomogeneous by TLC and HPLC. (ESI+): calc (M+H)⁺: 767.34, found: 767.03.¹H NMR (300 MHz, CD₃CN) δ 8.63 (s, 1H), 8.07 (s, 1H), 7.44-7.36 (m, 2H),7.33-7.18 (m, 7H), 6.84-6.72 (m, 4H), 6.02 (d, J=3.2 Hz, 1H), 4.74 (td,J=6.2, 0.9 Hz, 2H), 4.71-4.64 (m, 2H), 4.14-4.06 (m, 1H), 3.88-3.73 (m,8H), 3.57-3.41 (m, 4H), 3.32-3.23 (m, 3H), 3.08 (t, J=6.2 Hz, 2H), 2.77(dt, J=13.7, 6.9 Hz, 1H), 1.14 (dd, J=6.9, 1.2 Hz, 6H).

Synthesis of Compound 216

Compound 216: Using a procedure analogous to that described for compound208 and substituting compound 215 for compound 203, pure compound 216was obtained as a colorless solid foam. ¹H NMR (399 MHz, CD₃CN) δ 8.41(s, 1H), 8.12 (s, 1H), 7.50-7.44 (m, 2H), 7.40-7.19 (m, 12H), 6.80 (dd,J=8.9, 5.2 Hz, 4H), 6.04 (d, J=5.7 Hz, 1H), 4.99 (t, J=5.5 Hz, 1H), 4.94(ddd, J=9.9, 5.2, 3.7 Hz, 1H), 4.75 (t, J=6.2 Hz, 2H), 4.29 (q, J=3.8Hz, 1H), 3.89-3.82 (m, 1H), 3.76-3.68 (m, 7H), 3.64 (ddd, J=6.8, 5.5,2.3 Hz, 1H), 3.50-3.46 (m, 2H), 3.42 (s, 2H), 3.38-3.28 (m, 1H), 3.21(s, 3H), 3.09 (t, J=6.2 Hz, 2H), 2.84 (dq, J=10.2, 7.3 Hz, 1H),2.70-2.58 (m, 1H), 1.76 (s, 3H), 1.46-1.27 (m, 2H), 1.12 (d, J=6.8 Hz,3H), 1.07 (d, J=6.8 Hz, 3H), 1.04-0.89 (m, 2H); ³¹P NMR (162 MHz, CD₃CN)δ 154.40.

Synthesis of Compound 217

Compound 217: Using a procedure analogous to that described for compound210 and substituting compound 215 for compound 209, pure compound 217was obtained as a colorless solid foam. ¹H NMR (399 MHz, CD₃CN) δ 8.56(s, 1H), 8.13 (s, 1H), 7.44 (dd, J=8.2, 1.5 Hz, 2H), 7.39-7.19 (m, 12H),6.81 (dd, J=10.4, 8.9 Hz, 4H), 6.06 (d, J=4.5 Hz, 1H), 4.96-4.86 (m,2H), 4.75 (t, J=6.2 Hz, 2H), 4.30 (td, J=5.1, 4.7, 2.4 Hz, 1H), 3.83(dt, J=11.2, 4.3 Hz, 1H), 3.79-3.70 (m, 7H), 3.67 (ddd, J=7.1, 5.2, 2.0Hz, 1H), 3.52-3.45 (m, 3H), 3.41 (dd, J=10.8, 2.7 Hz, 1H), 3.34-3.24 (m,1H), 3.21 (s, 3H), 3.08 (t, J=6.1 Hz, 2H), 2.89-2.72 (m, 2H), 1.68 (s,3H), 1.54-1.38 (m, 2H), 1.14 (dd, J=6.8, 5.4 Hz, 6H), 1.09-1.00 (m, 1H),0.95-0.83 (m, 1H); ³¹P NMR (162 MHz, CD₃CN) δ 154.93.

Synthesis of Compound 219

Compound 219: Using a procedure analogous to that described for compound208 and substituting compound 218 for compound 203, pure compound 219was obtained as a colorless solid foam. ¹H NMR (399 MHz, CDCl₃) δ 9.31(s, 1H), 8.72 (s, 1H), 8.26 (s, 1H), 8.07-7.98 (m, 2H), 7.60-7.53 (m,1H), 7.52-7.43 (m, 4H), 7.38-7.15 (m, 12H), 6.83-6.74 (m, 4H), 6.24 (d,J=5.2 Hz, 1H), 5.03-4.92 (m, 2H), 4.45 (q, J=3.8 Hz, 1H), 3.93 (dt,J=11.4, 4.2 Hz, 1H), 3.77 (ddd, J=7.5, 5.8, 3.2 Hz, 2H), 3.73 (s, 6H),3.60-3.49 (m, 3H), 3.47-3.37 (m, 2H), 3.26 (s, 3H), 3.02-2.92 (m, 1H),1.81 (s, 3H), 1.56-1.43 (m, 1H), 1.37 (dq, J=13.2, 6.5 Hz, 1H),1.22-1.09 (m, 1H), 0.96 (ddt, J=13.1, 7.9, 6.8 Hz, 1H); ³¹P NMR (162MHz, CDCl₃) δ 157.73.

Synthesis of Compound 220

Compound 220: Using a procedure analogous to that described for compound210 and substituting compound 218 for compound 209, pure compound 220was obtained as a colorless solid foam. ¹H NMR (399 MHz, CDCl₃) δ 9.26(s, 1H), 8.73 (s, 1H), 8.31 (s, 1H), 8.02 (d, J=7.6 Hz, 2H), 7.60-7.53(m, 1H), 7.52-7.40 (m, 4H), 7.39-7.13 (m, 12H), 6.84-6.76 (m, 4H), 6.24(d, J=4.5 Hz, 1H), 4.99 (dt, J=10.0, 5.0 Hz, 1H), 4.84 (t, J=4.7 Hz,1H), 4.46 (q, J=4.0 Hz, 1H), 3.91 (dt, J=11.1, 4.2 Hz, 1H), 3.85-3.69(m, 8H), 3.63-3.50 (m, 3H), 3.42 (dd, J=10.7, 4.0 Hz, 1H), 3.40-3.31 (m,1H), 3.27 (s, 3H), 3.00-2.89 (m, 1H), 1.82 (s, 3H), 1.58-1.38 (m, 2H),1.20-1.09 (m, 1H), 1.00 (ddt, J=12.3, 8.8, 5.9 Hz, 1H); ³¹P NMR (162MHz, CDCl₃) δ 158.98.

Synthesis of Compound 222

Compound 222: Using a procedure analogous to that described for compound208 and substituting compound 221 for compound 203, pure compound 222was obtained as a colorless solid foam. ¹H NMR (499 MHz, CDCl₃) δ 10.46(s, br, 1H), 8.62 (d, J=7.5 Hz, 1H), 7.42 (d, J=7.1 Hz, 2H), 7.36-7.21(m, 12H), 7.00 (d, J=7.5 Hz, 1H), 6.82 (dd, J=9.0, 7.1 Hz, 4H), 6.03 (s,1H), 4.77 (td, J=8.7, 4.8 Hz, 1H), 4.34 (dt, J=9.4, 2.3 Hz, 1H), 3.97(d, J=4.8 Hz, 1H), 3.78 (dd, J=6.7, 3.4 Hz, 1H), 3.74 (s, 3H), 3.71 (s,6H), 3.68-3.58 (m, 2H), 3.48-3.38 (m, 1H), 3.01 (p, J=7.8 Hz, 1H), 2.27(s, 3H), 1.72 (s, 3H), 1.49 (tt, J=12.3, 7.0 Hz, 1H), 1.40 (dq, J=13.2,6.6 Hz, 1H), 1.18 (dq, J=13.9, 7.0 Hz, 1H), 1.02-0.90 (m, 1H); ³¹P NMR(202 MHz, CDCl₃) δ 158.71.

Synthesis of Compound 223

Compound 223: Using a procedure analogous to that described for compound210 and substituting compound 221 for compound 209, pure compound 223was obtained as a colorless solid foam. ¹H NMR (500 MHz, CDCl₃) δ 9.76(s, 1H), 8.70 (d, J=7.4 Hz, 1H), 7.48-7.41 (m, 2H), 7.41-7.18 (m, 12H),7.06 (d, J=7.4 Hz, 1H), 6.87 (dd, J=8.9, 1.9 Hz, 4H), 6.00 (s, 1H), 4.80(td, J=9.2, 4.7 Hz, 1H), 4.33 (d, J=9.5 Hz, 1H), 3.84 (d, J=4.7 Hz, 1H),3.81 (s, 3H), 3.80 (s, 3H), 3.72 (td, J=6.6, 3.4 Hz, 1H), 3.70-3.62 (m,4H), 3.53 (dd, J=11.3, 2.2 Hz, 1H), 3.34 (tdd, J=10.7, 7.8, 4.9 Hz, 1H),2.96 (dq, J=10.3, 7.6 Hz, 1H), 2.25 (s, 3H), 1.83 (s, 3H), 1.56-1.48 (m,1H), 1.43 (dq, J=13.1, 6.4 Hz, 1H), 1.24-1.16 (m, 1H), 1.00-0.91 (m,1H). ³¹P NMR (202 MHz, CDCl₃) δ 157.17.

Synthesis of Compound 225

Compound 225: Using a procedure analogous to that described for compound208 and substituting compound 224 for compound 203, pure compound 225was obtained as a colorless solid foam. ¹H NMR (500 MHz, CDCl₃) δ 9.76(s, br, 1H), 8.70 (d, J=7.4 Hz, 1H), 7.48-7.41 (m, 2H), 7.41-7.18 (m,12H), 7.06 (d, J=7.4 Hz, 1H), 6.87 (dd, J=8.9, 1.9 Hz, 4H), 6.00 (s,1H), 4.80 (td, J=9.2, 4.7 Hz, 1H), 4.33 (d, J=9.5 Hz, 1H), 3.84 (d,J=4.7 Hz, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.72 (td, J=6.6, 3.4 Hz, 1H),3.69-3.62 (m, 4H), 3.53 (dd, J=11.3, 2.2 Hz, 1H), 3.34 (tdd, J=10.7,7.8, 4.9 Hz, 1H), 2.96 (dq, J=10.3, 7.6 Hz, 1H), 2.25 (s, 3H), 1.83 (s,3H), 1.56-1.48 (m, 1H), 1.43 (dq, J=13.1, 6.4 Hz, 1H), 1.24-1.16 (m,1H), 1.00-0.91 (m, 1H); ³¹P NMR (202 MHz, CDCl₃) δ 158.97.

Synthesis of Compound 226

Compound 226: Using a procedure analogous to that described for compound210 and substituting compound 224 for compound 209, pure compound 226was obtained as a colorless solid foam. ¹H NMR (499 MHz, CDCl₃) δ9.8-9.0 (br, 1H), 8.09 (d, J=8.2 Hz, 1H), 7.40 (d, J=7.4 Hz, 2H),7.36-7.19 (m, 12H), 6.80 (dd, J=11.4, 8.8 Hz, 4H), 6.02 (d, J=2.2 Hz,1H), 5.22 (d, J=8.1 Hz, 1H), 4.82 (td, J=8.1, 4.9 Hz, 1H), 4.27 (d,J=7.4 Hz, 1H), 3.96 (dd, J=5.0, 2.2 Hz, 1H), 3.81-3.76 (m, 1H), 3.74 (s,3H), 3.70 (s, 3H), 3.62 (s, 3H), 3.61-3.54 (m, 2H), 3.50-3.41 (m, 1H),3.04 (dtd, J=10.2, 8.0, 6.3 Hz, 1H), 1.75 (s, 3H), 1.56-1.47 (m, 1H),1.44-1.35 (m, 1H), 1.26-1.18 (m, 1H), 0.95 (dq, J=12.3, 7.5 Hz, 1H); ³¹PNMR (202 MHz, CDCl₃) δ 158.96.

Synthesis of Compound 228

Compound 228: Using a procedure analogous to that described for compound208 and substituting compound 227 for compound 203, pure compound 228was obtained as a colorless solid foam. ³¹P NMR (202 MHz, CDCl₃) δ158.16.

Synthesis of Compound 229

Compound 229: Using a procedure analogous to that described for compound210 and substituting compound 227 for compound 209, pure compound 229was obtained as a colorless solid foam. ³¹P NMR (202 MHz, CDCl₃) δ158.84.

Synthesis of Compound 231

Compound 231: Using a procedure analogous to that described for compound208 and substituting compound 230 for compound 203, pure compound 231was obtained as a colorless solid foam. ¹H NMR (499 MHz, CDCl₃) δ9.6-9.3 (s, 1H), 8.73 (s, 1H), 8.32 (s, 1H), 7.50-7.41 (m, 2H),7.39-7.16 (m, 14H), 7.10-7.01 (m, 3H), 6.82 (d, J=8.8 Hz, 4H), 6.21 (d,J=4.1 Hz, 1H), 4.96 (dt, J=10.1, 5.1 Hz, 1H), 4.86 (s, 2H), 4.54 (t,J=4.5 Hz, 1H), 4.43 (q, J=3.9 Hz, 1H), 3.81-3.75 (m, 7H), 3.62 (dd,J=10.8, 3.3 Hz, 1H), 3.54 (s, 3H), 3.46-3.42 (m, 1H), 3.41-3.33 (m, 1H),3.01-2.93 (m, 1H), 1.82 (s, 3H), 1.56-1.47 (m, 1H), 1.44 (dq, J=13.2,6.5 Hz, 1H), 1.22-1.11 (m, 1H), 1.03-0.94 (m, 1H); ³¹P NMR (202 MHz,CDCl₃) δ 158.11.

Synthesis of Compound 232

Compound 232: Using a procedure analogous to that described for compound210 and substituting compound 230 for compound 209, pure compound 232was obtained as a colorless solid foam. ¹H NMR (499 MHz, CDCl₃) δ9.8-9.2 (br, 1H), 8.71 (s, 1H), 8.23 (s, 1H), 7.49-7.43 (m, 2H),7.40-7.15 (m, 14H), 7.09-7.02 (m, 3H), 6.79 (d, J=8.9 Hz, 4H), 6.20 (d,J=5.6 Hz, 1H), 4.93 (ddd, J=9.3, 5.0, 3.8 Hz, 1H), 4.86 (s, 2H), 4.73(t, J=5.3 Hz, 1H), 4.43 (q, J=3.9 Hz, 1H), 3.79-3.75 (m, 1H), 3.74 (s,3H), 3.74 (s, 3H), 3.57 (dt, J=6.8, 3.8 Hz, 1H), 3.52 (s, 3H), 3.46-3.36(m, 2H), 2.99 (dtd, J=10.1, 7.9, 6.5 Hz, 1H), 1.80 (s, 3H), 1.54-1.45(m, 1H), 1.39-1.31 (m, 1H), 1.23-1.13 (m, 1H), 0.99-0.90 (m, 1H); ³¹PNMR (202 MHz, CDCl₃) δ 158.13.

Synthesis of Compound 234

Compound 234: Using a procedure analogous to that described for compound208 and substituting compound 233 for compound 203, pure compound 234was obtained as a colorless solid foam.

Synthesis of Compound 235

Compound 235: Using a procedure analogous to that described for compound210 and substituting compound 233 for compound 209, pure compound 235was obtained as a colorless solid foam. ³¹P NMR (202 MHz, CDCl₃) δ158.75.

Synthesis of Compound 237

Compound 237: Using a procedure analogous to that described for compound208 and substituting compound 236 for compound 203, pure compound 237was obtained as a colorless solid foam. ³¹P NMR (202 MHz, Chloroform-d)δ 159.51 (d, J_(P-F)=9.5 Hz).

Synthesis of Compound 238

Compound 238: Using a procedure analogous to that described for compound210 and substituting compound 236 for compound 209, pure compound 238was obtained as a colorless solid foam. ³¹P NMR (202 MHz, Chloroform-d)δ 159.48.

Synthesis of Compound 240

Compound 240: Using a procedure analogous to that described for compound208 and substituting compound 239 for compound 203, pure compound 240was obtained as a colorless solid foam. ³¹P NMR (202 MHz, CDCl₃) δ160.20 (d, J_(P-F)=11.0 Hz).

Synthesis of Compound 241

Compound 241: Using a procedure analogous to that described for compound210 and substituting compound 239 for compound 209, pure compound 241was obtained as a colorless solid foam. ³¹P NMR (202 MHz, CDCl₃) δ159.66 (d, J_(P-F)=8.4 Hz).

Synthesis of Compound 243

Compound 243: Using a procedure analogous to that described for compound208 and substituting compound 242 for compound 203, pure compound 243was obtained as a colorless solid foam. ³¹P NMR (202 MHz, CDCl₃) δ160.20 (d, J=11.0 Hz).

Synthesis of Compound 244

Compound 244: Using a procedure analogous to that described for compound210 and substituting compound 242 for compound 209, pure compound 244was obtained as a colorless solid foam. ³¹P NMR (202 MHz, CDCl₃) δ156.52 (d, J_(P-F)=8.5 Hz).

Synthesis of Compound 246

Compound 246: Using a procedure analogous to that described for compound208 and substituting compound 245 for compound 203, pure compound 246 isobtained as a colorless solid foam.

Synthesis of Compound 247

Compound 247: Using a procedure analogous to that described for compound210 and substituting compound 245 for compound 209, pure compound 247 isobtained as a colorless solid foam.

Synthesis of Compound 249

Compound 249: Using a procedure analogous to that described for compound208 and substituting compound 248 for compound 203, pure compound 249 isobtained as a colorless solid foam.

Synthesis of Compound 250

Compound 250: Using a procedure analogous to that described for compound210 and substituting compound 248 for compound 209, pure compound 250 isobtained as a colorless solid foam.

Synthesis of Compound 252

Compound 252: Using a procedure analogous to that described for compound208 and substituting compound 251 for compound 203, pure compound 252 isobtained as a colorless solid foam.

Synthesis of Compound 253

Compound 253: Using a procedure analogous to that described for compound210 and substituting compound 251 for compound 209, pure compound 253 isobtained as a colorless solid foam.

Synthesis of Compound 255

Compound 255: Using a procedure analogous to that described for compound208 and substituting compound 254 for compound 203, pure compound 255 isobtained as a colorless solid foam.

Synthesis of Compound 256

Compound 256: Using a procedure analogous to that described for compound210 and substituting compound 254 for compound 209, pure compound 256 isobtained as a colorless solid foam.

Synthesis of Compound 258

Compound 258: Using a procedure analogous to that described for compound208 and substituting compound 257 for compound 203, pure compound 258 isobtained as a colorless solid foam.

Synthesis of Compound 259

Compound 259: Using a procedure analogous to that described for compound210 and substituting compound 257 for compound 209, pure compound 259 isobtained as a colorless solid foam.

Examples 2-16

Summarized below in Table E-1 is the synthetic procedure for Examples2-16 on the DNA/RNA Synthesizer ABI-394.

TABLE E-1 Summary for Oligonucleotide Synthesis on a DNA/RNA SynthesizerABI-394 Used for the Synthesis of Examples 2-16. delivery time (sec)wait time (sec) step reaction reagent 1 μmol 10 μmol 1 μmol 10 μmol 1detritylation 3% TCA in DCM 3 + 60 + 3 + 90 + N.A. N.A. 10 10 2 coupling0.15 M phosphoramidite in 5 + 4 10 + 6 30 + 600 30 + 600 ACN + 1.2 MCMPT in ACN 3 capping 1 5% Pac₂O in THF/2,6-lutidine 20 30 60 60 4capping 2 5% Pac₂O in THF/2,6-lutidine + 20 30 60 60 16% NMI in THF 5sulfurization 0.3 M S-cyanoethyl- 10 + 4 × 2 15 + 4 × 4 300 + 3 × 300 +3 × MethylThioSulfonate 150 + 600 150 + 600

Examples 2-9: The oligonucleotides containing stereodefinedphosphorothioate diester internucleotidic linkages were synthesized onan ABI-394 DNA/RNA synthesizer according to the cycle summarized inTable E-1 using a 10 μmol synthesis column and 6.5 μmol of succinyllinked dC on CPG. The synthesis cycle was performed with removal of theterminal 5′-O-DMTr group (DMT Off). The solid support was washed withdry ACN and dried under a flux of argon. The dry solid support was thentreated with 5 mL of anhydrous 1 M solution of1,8-diazabicycloundec-7-ene (DBU) in dry ACN-trimethylsilylchloride—16:1 (v/v) for 10 min at r.t., during which time the solutionwas slowly pushed through the column by means of a plastic luer syringefixed to the outlet of the column. The support was then washed with dryACN and dried under vacuum. The dry CPG was placed in a plastic vial andthen treated with 3 mL of 28% aqueous ammonia for a period of 18 h atr.t. The solvents were evaporated to dryness, the residue wasre-suspended in 10% aqueous DMSO, and the solid support was filteredoff. The crude product was purified by anion exchange preparative HPLC(Gradient of 0.25 to 1.75 M NaCl in 20 mM NaOH). The fractions havingpurity above 95% were pooled, concentrated and desalted by reverse-phaseHPLC (Gradient of 0 to 80% ACN). The final desalted product waslyophilized from water.

Examples 10-16: The oligonucleotides containing stereodefinedphosphorothioate diester internucleotidic linkages were synthesized onan ABI-394 DNA/RNA synthesizer according to the cycle summarized inTable E-1 using 1 μmol synthesis column and 3 μmol of succinyl linked dCon CPG. The synthesis cycle was performed with removal of the terminal5′-O-DMTr group (DMT Off). The solid support was washed with dry ACN anddried under a flux of argon. The dry solid support was then treated with5 mL of anhydrous 1 M solution of 1,5-diazabicyclo(4.3.0) non-5-ene(DBN) in dry ACN-trimethylsilyl chloride—16:1 (v/v) for 10 min at r.t.The DBN solution was slowly pushed through the column by means of aplastic luer syringe fixed to the outlet of the column. The support wasthen washed with dry ACN and dried under vacuum. The dry CPG was placedin a plastic vial and was treated with 2 mL of 28% aqueous ammonia for aperiod of 18 h at r.t. The solvents were evaporated to dryness, theresidue was re-suspended in 10% aqueous DMSO, and the solid support wasfiltered off.

Purification and Desalting of Examples 2-16:

The crude product was purified by Waters 2525 BGM HPLC system, equippedwith a 2487 dual wavelength detector and with FCO and Flex inject. AnAP-1 glass column from Waters, 10×200 mm was filled with Source 15QSupport (GE Healthcare, Part no. 17-0947-01) and was used with flow rateof 4 mL/min. Column was heated during all the purifications using aTL600 mobile phase heater and TL150 Temperature controller (TimberlineInstruments) set at 75° C. Buffer A: 20 mM NaOH and Buffer B: 20 mMNaOH, 2.5 M NaCl were used with step gradients starting from 30% B to70% B. The fractions having purity above 95% were pooled, concentratedand desalted on the same HPLC system by reverse-phase column (XBridgeSemi Prep, 250×10 mm, C₁₈, 5 μm) with a gradient of water to 80% ACN and4 mL/min flow rate. The final desalted product was concentrated inspeedvac followed by lyophilization from water.

HPLC analysis of purified oligonucleotides: Quality of oligonucleotideswas determined using DNA Pac 100 (10×250 mm) and using the followingconditions:

Buffer A: 10 mM Tris HCl, 50% HCl, pH=8.0Buffer B: A+0.8 M NaClO₄, pH=8.0Column temperature: 60° C.Gradient method:

Time Flow % A % B Curve 1 0.01 1.00 85.0 15.0 2 3.00 1.00 85.0 15.0 1 323.00 1.00 40.0 60.0 6 4 25.00 1.00 5.0 95.0 6 5 25.50 1.00 85.0 15.0 66 30.00 1.00 85.0 15.0 1LCMS analysis method:

Eluent A: 15 mM TEA, 400 mM HFIP, Water Eluent B: 50:50 BufferA/Methanol Column: UPLC@OST C₁₈ 1.7 μm, 2.1×500 mm

Column temperature=50° C.Gradient method:

Time Flow % A % B Curve 0 0.2 95 5 10 0.2 35 65 6 12 0.2 5 95 6 12.5 0.295 5 6 15 0.2 95 5 1

Example 2. Synthesis of Oligonucleotide 101(All-(Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC]) (SEQ ID NO: 106)

Oligonucleotide 101 was synthesized as described above. RT in IEX-HPLC:14.70 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6310.4.

Example 3. Synthesis of Oligonucleotide 102(All-(Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC]) (SEQ ID NO: 106)

Oligonucleotide 102 was synthesized as described above. RT in IEX-HPLC:15.49 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6310.2.

Example 4. Synthesis of Oligonucleotide 103 ((Rp, Rp, Rp, Rp, Rp, Sp,Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)(5R-9S-5R))

Oligonucleotide 103 was synthesized as described above. RT in IEX-HPLC:15.10 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6310.3.

Example 5. Synthesis of Oligonucleotide 104 ((Sp, Sp, Sp, Sp, Sp, Rp,Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp,Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)(5S-9R-5S))

Oligonucleotide 104 was synthesized as described above. RT in IEX-HPLC:15.04 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6307.2.

Example 6. Synthesis of Oligonucleotide 105 ((Sp, Rp, Rp, Rp, Rp, Rp,Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp,Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)(1S-17R-1S))

Oligonucleotide 105 was synthesized as described above. RT in IEX-HPLC:14.75 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6310.2.

Example 7. Synthesis of Oligonucleotide 106 ((Rp, Sp, Sp, Sp, Sp, Sp,Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)(1R-17S-1R))

Oligonucleotide 106 was synthesized as described above. RT in IEX-HPLC:15.43 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6309.6.

Example 8. Synthesis of Oligonucleotide 107 ((Rp, Sp, Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)((R/S)₉R))

Oligonucleotide 107 was synthesized as described above. RT in IEX-HPLC:15.02 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6310.7.

Example 9. Synthesis of Oligonucleotide 108 ((Sp, Rp, Sp, Rp, Sp, Rp,Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)((S/R)₉S))

Oligonucleotide 108 was synthesized as described above. RT in IEX-HPLC:15.10 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6307.9.

Example 10. Synthesis of Oligonucleotide 109 ((Sp, Sp, Sp, Rp, Rp, Rp,Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp,Sp)d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)(3S-13R-3S))

Oligonucleotide 109 was synthesized as described above. RT in IEX-HPLC:14.91 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6309.5.

Example 11. Synthesis of Oligonucleotide 110 ((Rp, Rp, Rp, Sp, Sp, Sp,Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp,RP)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)(3R-13S-3R))

Oligonucleotide 110 was synthesized as described above. RT in IEX-HPLC:15.24 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6309.3.

Example 12. Synthesis of Oligonucleotide 111 ((Sp, Sp, Sp, Sp, Sp, Sp,Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)((18S/R¹⁹))

Oligonucleotide 111 was synthesized as described above. RT in IEX-HPLC:15.69 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6309.4.

Example 13. Synthesis of Oligonucleotide 113 ((Sp, Rp, Sp, Sp, Sp, Sp,Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp,Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)(18S/R²))

Oligonucleotide 113 was synthesized as described above. RT in IEX-HPLC:15.72 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6311.0.

Example 14. Synthesis of Oligonucleotide 114 ((Rp, Rp, Sp, Rp, Rp, Sp,Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp,Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)((RRS)₆-R))

Oligonucleotide 114 was synthesized as described above. RT in IEX-HPLC:14.14 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6313.7.

Example 15. Synthesis of Oligonucleotide 115 ((Sp, Rp, Rp, Sp, Rp, Rp,Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp,Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)(S-(RRS)₆))

Oligonucleotide 115 was synthesized as described above. RT in IEX-HPLC:14.30 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6313.7.

Example 16. Synthesis of Oligonucleotide 116 ((Rp, Sp, Rp, Rp, Sp, Rp,Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp,Rp)d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (SEQ ID NO: 106)(RS-(RRS)₅-RR))

Oligonucleotide 116 was synthesized as described above. RT in IEX-HPLC:14.17 min. UPLC/ESI-MS: Calcd for C₁₉₁H₂₄₆N₆₇O₁₀₂P₁₉S₁₉: 6310.2; Found:6312.4.

Results of Examples 2-16 are summarized in Table E-2, below:

TABLE E-2 Summary of Examples 2-16. Oli- Descrip- gonu- SEQ tion of RT-cleo- ID stereo- IEX tide NO: 5′-sequence-3′ chemistry (min) 101 106d[GsCsCsTsCsAsGsTsCsT All-(Rp) 14.70 sGsCsTsTsCsGsCsAsCsC] 102 106d[GsCsCsTsCsAsGsTsCsT All-(Sp) 15.49 sGsCsTsTsCsGsCsAsCsC] 103 106d[GsCsCsTsCsAsGsTsCsT 5R-9S-5R 15.10 sGsCsTsTsCsGsCsAsCsC] 104 106d[GsCsCsTsCsAsGsTsCsT 5S-9R-5S 15.04 sGsCsTsTsCsGsCsAsCsC] 105 106d[GsCsCsTsCsAsGsTsCsT 1S-17R-1S 14.75 sGsCsTsTsCsGsCsAsCsC] 106 106d[GsCsCsTsCsAsGsTsCsT 1R-17S-1R 15.43 sGsCsTsTsCsGsCsAsCsC] 107 106d[GsCsCsTsCsAsGsTsCsT (R/S)₉R 15.02 sGsCsTsTsCsGsCsAsCsC] 108 106d[GsCsCsTsCsAsGsTsCsT (S/R)₉S 15.10 sGsCsTsTsCsGsCsAsCsC] 109 106d[GsCsCsTsCsAsGsTsCsT 3S-13R-3S 14.91 sGsCsTsTsCsGsCsAsCsC] 110 106d[GsCsCsTsCsAsGsTsCsT 3R-13S-3R 15.24 sGsCsTsTsCsGsCsAsCsC] 111 106d[GsCsCsTsCsAsGsTsCsT 18S/R¹⁹ 15.69 sGsCsTsTsCsGsCsAsCsC] 113 106d[GsCsCsTsCsAsGsTsCsT 18S/R² 15.72 sGsCsTsTsCsGsCsAsCsC] 114 106d[GsCsCsTsCsAsGsTsCsT (RRS)₆-R 14.14 sGsCsTsTsCsGsCsAsCsC] 115 106d[GsCsCsTsCsAsGsTsCsT S-(RRS)₆ 14.30 sGsCsTsTsCsGsCsAsCsC] 116 106d[GsCsCsTsCsAsGsTsCsT RS-(RRS)₅- 14.17 sGsCsTsTsCsGsCsAsCsC] RR

Example 17. Synthesis of Control Oligonucleotides

The control oligonucleotides (see Table E-3) were synthesized using thestandard chemistry methods for automated solid-phase oligonucleotidesynthesis (Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311). More specifically, stereorandom DNA was synthesized usingstandard DNA phosphoramidites (ChemGenes Co.), ethylthiotetrazole (ETT,Muang et al., Tetrahedron Lett., 2004, 45, 6497-6499) as the activator(Glen Research) andN,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)methanimidamide (DDTT,AM Chemicals) as the sulfurizing reagent (Guzaev, A.; Tetrahedron Lett.,2011, 52, 434-437). The phosphoramidite coupling time was 2 min and thesulfurization time was 10 min. The oligonucleotide was deprotected andpurified using standard methods. DNA phosphodiester was synthesizedusing standard DNA phosphoramidites, ETT as the activator andiodine/pyridine/water as the oxidizing reagent. 2′-O-Methoxyethyl (MOE)DNA was synthesized using in-house prepared 2′-O-Methoxyethyl (MOE)phosphoramidites (Martin, P.; Helv. Chim. Acta. 1995, 78, 486-504; Ross,B.; Song, Q.; 2004, US patent publication No. 20040082775), ETT as theactivator and DDTT as the sulfurizing reagent. The times for couplingwere 10 min and the times for sulfurization were 10 min. RNA wassynthesized using standard RNA phosphoramidites (ChemGenes Co.), ETT asthe activator and iodine/pyridine/water as the oxidizing reagent. Thecoupling times were 10 min.

All oligonucleotides were deprotected and purified using standardmethods.

Purification of RNA (Oligonucleotide 117):

Waters 2525 BGM, 2487 Dual wavelength detector equipped with FCO andFlex injectorBuffer A: 20 mM Sodium Phosphate pH=8.5Buffer B: 20 mM Sodium Phosphate, 1 M NaBr pH=8.5Column: AP-1 glass column from Waters, 10×200 mm, filled with SuperQ-5PW (20), TSK Gel (Anion Exchange) from TOSOHColumn Temperature: 70° C. (Timberline Instruments, TL600 mobile phaseheater and TL150 Temperature controller)Gradient used:

Time Flow (mL/min) % A % B Curve Initial 100 0 10 4 100 0 1 25 4 80 20 6115 4 55 45 6 125 4 0 100 6 130 4 100 0 6 140 4 100 0 1

As shown in FIG. 1, the chirally controlled phosphorothioate diester20-mer oligonucleotide (All-(Rp), Oligonucleotide 101, FIG. 1, A) has adifferent retention time than that of the phosphorothioate diester20-mer standard stereorandom oligonucleotide (Oligonucleotide 118, FIG.1, C) and has a sharper peak. One of skill in the art understands thatduring purification of the stereorandom oligonucleotide 108, it islikely that most of the all-(Rp) oligonucleotide (101, present in anapproximately ½¹⁹ fraction of the mixture stereorandom oligonucleotide108) would be lost.

Results of Example 17 are summarized in Table E-3, below.

TABLE E-3 Summary of Example 17. Oligonu- SEQ ID RT cleotide NO:5′-sequence-3′ Description (min) 117 114 GGUGCGAAGCAGAC RNA  5.10 UGAGGC118 106 d[GsCsCsTsCsAs stereo- 15.04 GsTsCsTsGsCsTs randomTsCsGsCsAsCsC] 119 106 d[GCCTCAGTC DNA  6.90 TGC TTC GCACC] 120 106(Gs5mCs5mCsTs5 stereo- 15.49 mCs)_(MOE) random d[AsGsTs5mCsTsGs5mCsTsTs5mCs] (Gs5mCsAs5mCs5 mC)_(MOE) 121 115 d[GsAsTsGsCsCsscrambled 15.09 TsCsTsCsCsTsAs CsGsCsGsCsCsT]

Procedures for Examples 18-21: The oligonucleotides containingstereodefined morpholinoethyl phosphorothioate triester internucleotidiclinkages were synthesized on an ABI-394 DNA/RNA synthesizer according tothe cycle summarized in Table E-4 using 1 μmol synthesis column and 0.8μmol of oxalyl linked dC on HCP. The synthesis cycle was performed withremoval of the terminal 5′-O-DMTr group (DMT Off). The solid support waswashed with dry ACN and dried under a flux of argon. The dry HCP wasplaced in a plastic vial and was treated with 1 mL of dry propylamine indry pyridine (in a 1:4 ratio) for a period of 18 h at r.t. The solventswere then evaporated and the residue was re-suspended with ˜pH 1.5aqueous solution containing 10% DMSO and the HCP support was filteredoff. The crude product was purified by reverse phase preparative HPLC.The fractions having purity above 95% were pooled, concentrated anddesalted by reverse-phase HPLC (Gradient of 0 to 80% ACN). The finaldesalted product was lyophilized from water.

TABLE E-4 Summary for Oligonucleotide Synthesis on a DNA/RNA SynthesizerABI-394 Used for the Synthesis of Examples 18-21. delivery time waittime step reaction reagent (sec) (sec) 1 detritylation 3% TCA in DCM 3 +60 + 10 N.A. 2 coupling 0.15 M phosphoramidite in ACN + 1.2 M CMPT 5 + 430 + 600 in ACN 3 capping 1 5% Pac₂O in THF/2,6-lutidine 20 60 4 capping2 5% Pac₂O in THF/2,6-lutidine + 16% NMI in THF 20 60 5 sulfurization0.3 M S-Morpholinoethyl-ToluylThioSulfonate 10 + 4 × 2 300 + 3 × 150 +600

General Purification Method for Examples 18-21:

Buffer A: 20 mM Phosphate pH=6.0 (adjusted with phosphoric acid)

Buffer B: ACN

Column: XBridge Prep C₁₈, 5 μm, C₁₈, 250×10 mm, Part #186003256Buffer heater set temperature=50° C.Signal monitored at 254 and 280 nmGradient used:

Time Flow (ml/min) % A % B Curve Initial 99 1 5 4 99 1 1 10 4 77 23 6 604 70 30 6 65 4 20 80 6 70 4 20 80 6 71 4 99 1 6 80 4 99 1 1

Analytical HPLC methods for oligonucleotides.

HPLC Method 1:

Buffer A: 20 mM Phosphate pH=6.0 (adjusted with phosphoric acid)

Buffer B: ACN

Column: XBridge C₁₈, 3.5 μm, C₁₈, 4.6×50 mm, Part #186003034Buffer heater set temperature=35° C.Signal monitored at 254 and 280 nmGradient used:

Time Flow (ml/min) % A % B Curve Initial 95 5 3 1 95 5 1 23 1 60 40 6 251 40 60 6 25.5 1 95 5 6 30 1 95 5 1

HPLC Method 2: Buffer A: 50 mM TEAA, pH 7.8 Buffer B: ACN

Column: XBridge C₁₈, 3.5 μm, C₁₈, 4.6×50 mm, Part #186003034Buffer heater set temperature=60° C.Signal monitored at 254 and 280 nmGradient used:

Time Flow (ml/min) % A % B Curve Initial 99 1 2 1 99 1 1 22 1 65 35 6 251 5 95 6 25.5 1 5 95 6 30 1 99 1 1

HPLC Method 3:

Time Flow (ml/min) % A % B Curve Initial 85 15 2 1 85 15 1 20 1 60 40 622 1 5 95 6 25 1 5 95 6 25.5 1 85 15 6 30 1 85 15 1

HPLC Method 4:

Time Flow (ml/min) % A % B Curve Initial 85 15 2 1 85 15 1 20 1 40 60 622 1 5 95 6 25 1 5 95 6 25.5 1 85 15 6 30 1 85 15 1

Example 18. Synthesis of Oligonucleotide 122(All-(Rp)-d[Gs1Cs1Cs1Ts1Cs1As1GsTs1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C](SEQ ID NO: 106)

Oligonucleotide 122 was synthesized as described above. RT in RP-HPLC:(HPLC method 1): 15.2 min. UPLC/ESI-MS: Calcd for C₃₀₅H₄₅₅N₈₆O₁₂₁P₁₉S₁₉:8460.25; Found: 8462.0.

Example 19. Synthesis of Oligonucleotide 123 ((Sp, Rp, Rp, Rp, Rp, Rp,Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp,Sp)-d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C] (SEQID NO: 106) ((1S-17R-1S)

Oligonucleotide 123 was synthesized as described above. RT in RP-HPLC(HPLC method 1): 16.2 min. UPLC/ESI-MS: Calcd for C₃₀₅H₄₅₅N₈₆O₁₂₁P₁₉S₁₉:8460.3; Found: 8461.5.

Example 20. Synthesis of Oligonucleotide 124(All-(Sp)-d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C](SEQ ID NO: 106))

Oligonucleotide 124 was synthesized as described above. RT in RP-HPLC(HPLC method 1): 18.3 min. UPLC/ESI-MS: Calcd for C₃₀₅H₄₅₅N₈₆O₁₂₁P₁₉S₁₉:8460.3; Found: 8461.8.

Example 21. Synthesis of Oligonucleotide 125 (All-(Rp)-d[5mCs1As1Ts1G](SEQ ID NO: 106))

Oligonucleotide 125 was synthesized as described above. RT in RP-HPLC(HPLC method 2): 16.32 min. UPLC/ESI-MS: Calcd for C₅₈H₈₅N₁₈O₂₂P₃S₃:1575.5; Found: 1575.2.

In Examples 22 and 42 the oligonucleotide containing stereodefinedmethoxyethyl phosphorothioate triester internucleotidic linkages wassynthesized on an ABI-394 DNA/RNA synthesizer according to the cyclesummarized in Table E-5 using 1 μmol synthesis column and 0.8 μmol ofoxalyl linked dC on HCP. The synthesis cycle was performed with removalof the terminal 5′-O-DMTr group (DMT Off). The solid support was washedwith dry ACN and dried under a flux of argon. The dry HCP was placed ina plastic vial and was treated with 1 mL of dry propylamine in drypyridine (in a 1:4 ratio) for a period of 18 h at r.t. The solvents werethen evaporated and the residue was re-suspended with ˜pH 1.5 aqueoussolution containing 10% DMSO and the HCP support was filtered off. Thecrude product is purified by reverse phase preparative HPLC (Gradient of5 to 65% ACN in 20 mM sodium phosphate buffer, pH=6.0). The fractionshaving purity above 95% are pooled, concentrated and desalted byreverse-phase HPLC (Gradient of 0 to 80% ACN). The final desaltedproduct is lyophilized from water.

TABLE E-5 Summary for Oligonucleotide Synthesis on a DNA/RNA SynthesizerABI-394 Used for the Synthesis of Examples 22 and 42. delivery time waittime step reaction reagent (sec) (sec) 1 detritylation 3% TCA in DCM 3 +60 + 10 N.A. 2 coupling 0.15 M phosphoramidite in ACN + 1.2 M CMPT 5 + 430 + 600 in ACN 3 capping 1 5% Pac₂O in THF/2,6-lutidine 20 60 4 capping2 5% Pac₂O in THF/2,6-lutidine + 16% NMI in THF 20 60 5 sulfurization0.3 M S-MOE Toluylthiosulfonate 10 + 4 × 2 300 + 3 × 150 + 600

Example 22. Synthesis of Oligonucleotide 126 (All-(Rp)-d[Cs2As2Gs2T]

Oligonucleotide 126 was synthesized as described above. RT in RP-HPLC(HPLC method 2): 16.23 min. UPLC/ESI-MS: Calcd for C₄₈H₆₈N₁₅O₂₂P₃S₃:1396.2; Found: 1395.2.

Example 23: The oligonucleotide containing stereodefinedN-methylpiperazino bulky ester phosphorothioate triesterinternucleotidic linkages was synthesized on an ABI-394 DNA/RNAsynthesizer according to the cycle summarized in Table E-6 using 1 μmolsynthesis column and 1.5 μmol of oxalyl linked dT on HCP. The synthesiscycle was performed with removal of the terminal 5′-O-DMTr group (DMTOff). The solid support was washed with dry ACN and dried under a fluxof argon. The dry HCP was placed in a plastic vial and was treated with1 mL of dry propylamine in dry pyridine (in a 1:4 ratio) for a period of18 h at r.t. The solvents were then evaporated and the residue wasre-suspended with ˜pH 1.5 aqueous solution containing 10% DMSO and theHCP support was filtered off. The crude product was purified by reversephase preparative HPLC (Gradient of 5 to 65% ACN in 20 mM sodiumphosphate buffer, pH=6.0). The fractions having purity above 95% werepooled, concentrated and desalted by reverse-phase HPLC (Gradient of 0to 80% ACN). The final desalted product was lyophilized from water.

TABLE E-6 Summary for Oligonucleotide Synthesis on a DNA/RNA SynthesizerABI-394 Used for the Synthesis of Example 23. delivery time wait timestep reaction reagent (sec) (sec) 1 detritylation 3% TCA in DCM 3 + 60 +10 N.A. 2 coupling 0.15 M phosphoramidite in ACN + 1.2 M CMPT in ACN 5 +4 30 + 600 3 capping 1 5% Pac₂O in THF/2,6-lutidine 20 60 4 capping 2 5%Pac₂O in THF/2,6-lutidine + 16% NMI in THF 20 60 5 sulfurization 0.3 MN-Methyl Piperazino ester PhenylThioSulfonate 10 + 4 × 2 300 + 3 × 150 +600

Example 23. Synthesis of Oligonucleotide 127 (All-(Rp)-d[Cs3As3Gs3T]

Oligonucleotide 127 was synthesized as described above. RT in RP-HPLC(HPLC method 2): 20.24 min. UPLC/ESI-MS: Calcd for C₇₈H₁₂₂N₂₁O₂₅P₃S₃:1943.1; Found: 1941.0.

Examples 24 and 43 The oligonucleotide containing stereodefinedmorpholino bulky ester phosphorothioate triester internucleotidiclinkages was synthesized on an ABI-394 DNA/RNA synthesizer according tothe cycle summarized in Table E-7 using 1 μmol synthesis column and 1.5μmol of oxalyl linked dT on HCP. The synthesis cycle was performed withremoval of the terminal 5′-O-DMTr group (DMT Off). The solid support waswashed with dry ACN and dried under a flux of argon. The dry HCP wasplaced in a plastic vial and was treated with 1 mL of dry propylamine indry pyridine (in a 1:4 ratio) for a period of 18 h at r.t. The solventswere then evaporated and the residue was re-suspended with ˜pH 1.5aqueous solution containing 10% DMSO and the HCP support was filteredoff. The crude product is purified by reverse phase preparative HPLC(Gradient of 5 to 65% ACN in 20 mM sodium phosphate buffer, pH=6.0). Thefractions having purity above 95% are pooled, concentrated and desaltedby reverse-phase HPLC (Gradient of 0 to 80% ACN). The final desaltedproduct is lyophilized from water.

TABLE E-7 Summary for Oligonucleotide Synthesis on a DNA/RNA SynthesizerABI-394 Used for the Synthesis of Examples 24 and 43. delivery time waittime step reaction reagent (sec) (sec) 1 detritylation 3% TCA in DCM 3 +60 + 10 N.A. 2 coupling 0.15 M phosphoramidite in ACN + 1.2 M CMPT inACN 5 + 4 30 + 600 3 capping 1 5% Pac₂O in THF/2,6-lutidine 20 60 4capping 2 5% Pac₂O in THF/2,6-lutidine + 16% NMI in THF 20 60 5sulfurization 0.3 M morpholino ester NitroPhenylThioSulfonate 10 + 4 × 2300 + 3 × 150 + 600

Example 24. Synthesis of Oligonucleotide 128 (All-(Sp)-d[Cs4As4Gs4T]

Oligonucleotide 128 was synthesized as described above. RT in RP-HPLC(HPLC method 3): 19.75 min. UPLC/ESI-MS: Calcd for C₇₅H₁₁₃N₁₈O₂₈P₃S₃:1902.9; Found: 1904.0.

Example 25: The oligonucleotide containing stereodefineddimethylaminoethyl phosphorothioate triester internucleotidic linkageswas synthesized on an ABI-394 DNA/RNA synthesizer according to the cyclesummarized in Table E-8 using 1 μmol synthesis column and 1.5 μmol ofoxalyl linked dT on HCP. The synthesis cycle was performed with removalof the terminal 5′-O-DMTr group (DMT Off). The solid support was washedwith dry ACN and dried under a flux of argon. The dry HCP was placed ina plastic vial and was treated with 1 mL of dry propylamine in drypyridine (in a 1:4 ratio) for a period of 18 h at r.t. The solvents werethen evaporated and the residue was re-suspended with ˜pH 1.5 aqueoussolution containing 10% DMSO and the HCP support was filtered off. Thecrude product was purified by reverse phase preparative HPLC (Gradientof 5 to 65% ACN in 20 mM sodium phosphate buffer, pH=6.0). The fractionshaving purity above 95% were pooled, concentrated and desalted byreverse-phase HPLC (Gradient of 0 to 80% ACN). The final desaltedproduct was lyophilized from water.

TABLE E-8 Summary for Oligonucleotide Synthesis on a DNA/RNA SynthesizerABI-394 Used for the Synthesis of Example 25. delivery time wait timestep reaction reagent (sec) (sec) 1 detritylation 3% TCA in DCM 3 + 60 +10 N.A. 2 coupling 0.15 M phosphoramidite in ACN + 1.2 M CMPT 5 + 4 30 +600 in ACN 3 capping 1 5% Pac₂O in THF/2,6-lutidine 20 60 4 capping 2 5%Pac₂O in THF/2,6-lutidine + 16% NMI in THF 20 60 5 sulfurization 0.3 Mdimethylaminoethyl-ToluylThioSulfonate 10 + 4 × 2 900 + 3 × 600 + 900

Example 25. Synthesis of Oligonucleotide 129 (All-(Sp)-d[Cs5As5Gs5T]

Oligonucleotide 129 was synthesized as described above. RT in RP-HPLC(HPLC method 2): 17.25 min. UPLC/ESI-MS: Calcd for C₅₁H₇₇N₁₈O₁₉P₃S₃:1435.4; Found: 1435.0.

Example 26: The oligonucleotide containing stereodefined dimethylalanineester phosphorothioate triester internucleotidic linkages wassynthesized on an ABI-394 DNA/RNA synthesizer according to the cyclesummarized in Table E-9 using 1 μmol synthesis column and 1.5 μmol ofoxalyl linked dT on HCP. The synthesis cycle was performed with removalof the terminal 5′-O-DMTr group (DMT Off). The solid support was washedwith dry ACN and dried under a flux of argon. The dry HCP was placed ina plastic vial and was treated with 1 mL of dry propylamine in drypyridine (in a 1:4 ratio) for a period of 18 h at r.t. The solvents werethen evaporated and the residue was re-suspended with ˜pH 1.5 aqueoussolution containing 10% DMSO and the HCP support was filtered off. Thecrude product is purified by reverse phase preparative HPLC (Gradient of5 to 65% ACN in 20 mM sodium phosphate buffer, pH=6.0). The fractionshaving purity above 95% are pooled, concentrated and desalted byreverse-phase HPLC (Gradient of 0 to 80% ACN). The final desaltedproduct is lyophilized from water.

TABLE E-9 Summary for Oligonucleotide Synthesis on a DNA/RNA SynthesizerABI-394 Used for the Synthesis of Example 26. delivery time wait timestep reaction reagent (sec) (sec) 1 detritylation 3% TCA in DCM 3 + 60 +10 N.A. 2 coupling 0.15 M phosphoramidite in ACN + 1.2 M CMPT in ACN 5 +4 30 + 600 3 capping 1 5% Pac₂O in THF/2,6-lutidine 20 60 4 capping 2 5%Pac₂O in THF/2,6-lutidine + 16% NMI in THF 20 60 5 sulfurization 0.3 Mdimethylalanine-Fmoc ester NitroPhenylThioSulfonate 10 + 4 × 2 900 + 3 ×600 + 900

Example 26. Synthesis of Oligonucleotide 130 (All-(Sp)-d[Cs6As6Gs6T]

Oligonucleotide 130 was synthesized as described above. RT in RP-HPLC(HPLC method 2): 16.45 min. UPLC/ESI-MS: Calcd for C₅₇H₈₃N₁₈O₂₅P₃S₃:1609.5; Found: 1609.6.

Examples 27 and 28: The oligonucleotide containing stereodefinedS-methyl phosphorothioate triester internucleotidic linkages wassynthesized on an ABI-394 DNA/RNA synthesizer according to the cyclesummarized in Table E-10 using 1 μmol synthesis column and 0.8 μmol ofoxalyl linked dC on HCP. The synthesis cycle was performed with removalof the terminal 5′-O-DMTr group (DMT Off). The solid support was washedwith dry ACN and dried under a flux of argon. The dry HCP was placed ina plastic vial and was treated with 1 mL of dry propylamine in drypyridine (in a 1:4 ratio) with 50% DMSO for a period of 18 h at r.t. Thesolvents were then evaporated and the residue was re-suspended with ˜pH1.5 aqueous solution containing 10% DMSO and the HCP support wasfiltered off. The crude product is purified by reverse phase preparativeHPLC (Gradient of 5 to 65% ACN in 20 mM sodium phosphate buffer,pH=6.0). The fractions having purity above 95% are pooled, concentratedand desalted by reverse-phase HPLC (Gradient of 0 to 80% ACN). The finaldesalted product is lyophilized from water.

TABLE E-10 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI-394 Used for the Synthesis of Examples 27 and 28.delivery time wait time step reaction reagent (sec) (sec) 1detritylation 3% TCA in DCM 3 + 60 + 10 N.A. 2 coupling 0.15Mphosphoramidite in 5 + 4 30 + 600 ACN + 1.2M CMPT in ACN 3 capping 1 5%Pac₂O in THF/2,6-lutidine 20 60 4 capping 2 5% Pac₂O inTHF/2,6-lutidine + 20 60 16% NMI in THF 5 sulfurization 0.3M S-methyl10 + 4 × 2 300 + 3 × 150 + NitroPhenylThioSulfonate 600 

Example 27. Synthesis of Oligonucleotide 131(All-(Rp)-d[Gs7Cs7Cs7Ts7Cs7As7Gs7Ts7Cs7Ts7Gs7Cs7Ts7Ts7Cs7Gs7Cs7As7Cs7C](SEQ ID NO: 106)

Oligonucleotide 131 was synthesized as described above. RT in RP-HPLC:27.65 min. UPLC/ESI-MS: Calcd for C₂₁₀H₂₈₄N₆₇O₁₀₂P₁₉S₁₉: 6576.71; Found:6575. 6.

Example 28. Synthesis of Oligonucleotide 132(All-(Sp)-d[Gs7Cs7Cs7Ts7Cs7As7Gs7Ts7Cs7Ts7Gs7Cs7Ts7Ts7Cs7Gs7Cs7As7Cs7C](SEQ ID NO: 106)

Oligonucleotide 132 was synthesized as described above. RT in RP-HPLC:32.65 min. UPLC/ESI-MS: Calcd for C₂₁₀H₂₈₄N₆₇O₁₀₂P₁₉S₁₉: 6576.71; Found:6574.8.

Synthesis of Chirally Controlled Oligonucleotides Comprising ModifiedNucleobases

As generally described above and herein, in some embodiments, thepresent invention provides chirally controlled oligonucleotidescomprising oligonucleotides other than A, T, C and G. In someembodiments, such chirally controlled oligonucleotides comprise5-methylcytosine (5mC). Non-limiting examples are presented in Examples21 and below.

Examples 29-41 were synthesized using the automated synthesis on ABI-394DNA/RNA synthesizer according to the synthetic cycle summarized on TableE-11, using 1 μmol synthesis column and 1.75 μmol of oxalyl linked dG onHCP. The synthesis cycle was performed with removal of the terminal5′-O-DMTr group (DMT Off). After completion of the automatedoligonucleotide synthesis, the HCP support was washed with dry ACN anddried under vacuum. The dry HCP was placed in a plastic vial and wastreated with 1 mL of dry propylamine in dry pyridine (in a 1:4 ratio)for a period of 18 h at r.t. The solvents were then evaporated and theresidue was re-suspended with ˜pH 1.5 aqueous solution containing 10%DMSO and the HCP support was filtered off. The crude product is purifiedby reverse phase preparative HPLC (According to the procedure describedbelow). The fractions having purity above 95% are pooled, concentratedand desalted by reverse-phase HPLC (Gradient of 0 to 30% ACN). The finaldesalted product is lyophilized from water.

TABLE E-11 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI-394 Used for the Synthesis of Examples 29-41. deliverytime step reaction reagent (sec) wait time (sec) 1 detritylation 3% TCAin DCM 3 + 60 + 10 N.A. 2 coupling 0.15M phosphoramidite in 5 + 4 30 +600 ACN + 1.2M CMPT in ACN 3 capping 1 5% Pac₂O in THF/2,6-lutidine 2060 4 capping 2 5% Pac₂O in THF/2,6-lutidine + 20 60 16% NMI in THF 5sulfurization 0.3M S-Morpholinoethyl 10 + 4 × 2 300 + 3 × 150 +ToluylThioSulfonate 600 

General Purification Method for Examples 29-41:

Buffer A: 20 mM Phosphate pH=6.0 (adjusted with phosphoric acid)

Buffer B: ACN

Column: XBridge Prep C₁₈, 5 μm, C₁₈, 250×10 mm, Part #186003256Buffer heater set temperature=50° C.Signal monitored at 254 and 280 nmGradient used:

Time Flow (ml/min) % A % B Curve Initial 99 1 5 4 99 1 1 10 4 77 23 6 604 70 30 6 65 4 20 80 6 70 4 20 80 6 71 4 99 1 6 80 4 99 1 1

Example 29. Synthesis of Oligonucleotide 135(All-(Rp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQ ID NO:108))

Oligonucleotide 135 was synthesized as described above. RT in RP-HPLC(HPLC method 1): 17.50 min. UPLC/ESI-MS: Calcd for C₁₈₆H₂₇₈N₅₁O₇₃P₁₁S₁₁:5090.0; Found: 5091.9.

Example 30. Synthesis of Oligonucleotide 136(All-(Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQ ID NO:108))

Oligonucleotide 136 was synthesized as described above. RT in RP-HPLC(HPLC method 1): 19.25 min. UPLC/ESI-MS: Calcd for C₁₈₆H₂₇₈N₅₁O₇₃P₁₁S₁₁:5090.0; Found: 5090.8.

Example 31. Synthesis of Oligonucleotide 137 ((Sp, Rp, Rp, Rp, Rp, Rp,Rp, Rp, Rp, Rp, Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQID NO: 108) (1S-9R-1S))

Oligonucleotide 137 was synthesized as described above. RT in RP-HPLC(HPLC method 1): 17.85 min. UPLC/ESI-MS: Calcd for C₁₈₆H₂₇₈N₅₁O₇₃P₁₁S₁₁:5090.0; Found: 5091.9.

Example 32. Synthesis of Oligonucleotide 138 ((Sp, Sp, Rp, Rp, Rp, Rp,Rp, Rp, Rp, Sp, Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQID NO: 108) (2S-7R-2S))

Oligonucleotide 138 was synthesized as described above. RT in RP-HPLC(HPLC method 1): 18.10 min. UPLC/ESI-MS: Calcd for C₁₈₆H₂₇₈N₅₁O₇₃P₁₁S₁₁:5090.0; Found: 5091.9.

Example 33. Synthesis of Oligonucleotide 139 ((Rp, Sp, Sp, Sp, Sp, Sp,Sp, Sp, Sp, Sp, Rp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQID NO: 108) (1R-9S-1R))

Oligonucleotide 139 was synthesized as described above. RT in RP-HPLC(HPLC method 1): 18.75 min. UPLC/ESI-MS: Calcd for C₁₈₆H₂₇₈N₅₁O₇₃P₁₁S₁₁:5090.0; Found: 5088.9.

Example 34. Synthesis of Oligonucleotide 140 ((Rp, Rp, Sp, Sp, Sp, Sp,Sp, Sp, Sp, Rp, Rp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQID NO: 108) (2R-7S-2R))

Oligonucleotide 140 was synthesized as described above. RT in RP-HPLC(HPLC method 1): 18.72 min. UPLC/ESI-MS: Calcd for C₁₈₆H₂₇₈N₅₁O₇₃P₁₁S₁₁:5090.0; Found: 5091.3.

Example 35. Synthesis of Oligonucleotide 141 ((Sp, Sp, Sp, Rp, Rp, Rp,Rp, Rp, Sp, Sp, Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQID NO: 108) (3S-5R-3S))

Oligonucleotide 141 was synthesized as described above. RT in RP-HPLC(HPLC method 1): 18.09 min. UPLC/ESI-MS: Calcd for C₁₈₆H₂₇₈N₅₁O₇₃P₁₁S₁₁:5090.0; Found: 5090.9.

Example 36. Synthesis of Oligonucleotide 142 ((Rp, Rp, Rp, Sp, Sp, Sp,Sp, Sp, Rp, Rp, Rp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQID NO: 108) (3R-5S-3R))

Oligonucleotide 142 was synthesized as described above. RT in RP-HPLC:18.35 min. UPLC/ESI-MS (HPLC method 1): Calcd for C₁₈₆H₂₇₈N₅₁O₇₃P₁₁S₁₁:5090.0; Found: 5088.9.

Example 37. Synthesis of Oligonucleotide 143 ((Sp, Sp, Rp, Sp, Sp, Rp,Sp, Sp, Rp, Sp, Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQID NO: 108) ((SSR)₃-SS))

Oligonucleotide 143 was synthesized as described above. RT in RP-HPLC(HPLC method 1): 18.48 min. UPLC/ESI-MS: Calcd for C₁₈₆H₂₇₈N₅₁O₇₃P₁₁S₁₁:5090.0; Found: 5092.0.

Example 38. Synthesis of Oligonucleotide 144 ((Rp, Rp, Sp, Rp, Rp, Sp,Rp, Rp, Sp, Rp, Rp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQID NO: 108) ((RRS)₃-RR))

Oligonucleotide 144 was synthesized as described above. RT in RP-HPLC(HPLC method 1): 18.02 min. UPLC/ESI-MS: Calcd for C₁₈₆H₂₇₈N₅₁O₇₃P₁₁S₁₁:5090.0; Found: 5091.4.

Example 39. Synthesis of Oligonucleotide 145(All-(Rp)-d[5mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1Gs15mC](SEQ ID NO: 108))

Oligonucleotide 145 was synthesized as described above. RT in RP-HPLC(HPLC method 1): 17.30 min. UPLC/ESI-MS: Calcd for C₂₃₄H₃₅₂N₆₂O₉₂P₁₄S₁₄:6388.3; Found: 6390.6.

Example 40. Synthesis of Oligonucleotide 146 (All-(Rp)-d[Gs15mCs1Ts1G])

Oligonucleotide 146 was synthesized as described above. RT in RP-HPLC(HPLC method 2): 15.89 min. UPLC/ESI-MS: Calcd for C₅₈H₈₅N₁₈O₂₃P₃S₃:1591.5; Found: 1590.8.

Example 41. Synthesis of Oligonucleotide 147 (All-(Rp)-d[5mCs1As1Gs1T])

Oligonucleotide 147 was synthesized as described above. RT in RP-HPLC(HPLC method 2): 16.30 min. UPLC/ESI-MS: Calcd for C₅₈H₈₅N₁₈O₂₂P₃S₃:1575.5; Found: 1575.2.

Example 42. Synthesis of Oligonucleotide 148(All-(Rp)-d[5mCs2As2Gs2Ts25mCs2Ts2Gs25mCs2Ts2Ts25mCs2G] (SEQ ID NO:108))

Oligonucleotide 148 was synthesized as described above, using thesulfurization reagent in Example 22. RT in RP-HPLC (HPLC method 3):16.51 min. UPLC/ESI-MS: Calcd for C₁₅₃H₂₂₃N₄₀O₇₃P₁₁S₁₁: 4484.1; Found:4483.0.

Example 43. Synthesis of Oligonucleotide 149(All-(Rp)-d[5mCs4As4Gs4Ts45mCs4Ts4Gs45mCs4Ts4Ts45mCs4G] (SEQ ID NO:108))

Oligonucleotide 149 was synthesized as described above, using thesulfurization reagent in Example 24. RT in RP-HPLC (HPLC method 4):17.87 min. UPLC/ESI-MS: Calcd for C₂₅₂H₃₈₈N₅₁O₉₅P₁₁S₁₁: 6345.6; Found:6346.5.

Synthesis of Chirally Controlled Chimeric Oligonucleotides ComprisingDifferent Internucleotidic Linkages

As generally described above and herein, in some embodiments, thepresent invention provides chirally controlled oligonucleotidecomprising different internucleotidic linkages. The non-limitingexamples below illustrate such chirally controlled oligonucleotides, andthe methods of synthesizing the same.

Examples 44-45 illustrate the synthesis of chirally controlled chimericoligonucleotide comprising different internucleotidic linkages. Theoligonucleotides containing mixed diastereomerically puremorpholinoethyl phosphorothioate triester/phosphorothioate diesterinternucleotidic linkages were synthesized on an ABI-394 DNA/RNAsynthesizer according to the cycle summarized in Table E-12 using 1 μmolsynthesis column and 1.5 μmol of oxalyl linked dT on HCP. As eachiterative cycle allows for a different sulfurization reagent to bereacted, two different thiosulfonates were used. The synthesis wasperformed with removal of the terminal 5′-O-DMTr group (DMT Off). Thesolid support was washed with dry ACN and dried under a flux of argon.The dry solid support was then treated with 5 mL of anhydrous 1 Msolution of 1,5-diazabicyclo(4.3.0)non-5-ene (DBN) in dryACN-trimethylsilyl chloride—16:1 (v/v) for 10 min at r.t. The DBNsolution was slowly pushed through the column by means of a plastic luersyringe fixed to the outlet of the column. The support was then washedwith dry ACN and dried under vacuum. The dry HCP was placed in a plasticvial and was treated with 1 mL of dry propylamine in dry pyridine (in a1:4 ratio) for a period of 18 h at r.t. The solvents were thenevaporated and the residue was re-suspended with ˜pH 1.5 aqueoussolution containing 10% DMSO and the HCP support was filtered off. Thecrude product is purified by reverse phase preparative HPLC (Gradient of5 to 65% ACN in 20 mM sodium phosphate buffer, pH=6.0). The fractionshaving purity above 95% are pooled, concentrated and desalted byreverse-phase HPLC (Gradient of 0 to 80% ACN). The final desaltedproduct is lyophilized from water.

TABLE E-12 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI-394 Used for the Synthesis of Examples 44-45. deliverytime step reaction reagent (sec) wait time (sec) 1 detritylation 3% TCAin DCM 3 + 60 + 10 N.A. 2 coupling 0.15M phosphoramidite in 5 + 4 30 +600 ACN + 1.2M CMPT in ACN 3 capping 1 5% Pac₂O in THF/2,6-lutidine 2060 4 capping 2 5% Pac₂O in THF/2,6-lutidine + 20 60 16% NMI in THF 5sulfurization 0.3M S-Morpholinoethyl 10 + 4 × 2 300 + 3 × 150 + 1Toluylthiosulfonate 600 

6 sulfurization 0.3M S-cyanoethyl 10 + 4 × 2 300 + 3 × 150 + 2Methylthiosulfonate 600 

Example 44. Synthesis of Oligonucleotide 150 (All-(Rp)-d[TsCs1AsT])

Oligonucleotide 150 was synthesized as described above. RT in RP-HPLC(HPLC method 2): 12.72 min. UPLC/ESI-MS: Calcd for C₄₅H₆₂N₁₃O₂₁P₃S₃:1310.2; Found: 1309.2

Example 45. Synthesis of Oligonucleotide 151 (All-(Sp)-d[Cs1AsGs1T])

Oligonucleotide 151 was synthesized as described above. RT in RP-HPLC(HPLC method 2): 14.71 min. UPLC/ESI-MS: Calcd for C₅₁H₇₂N₁₇O₂₁P₃S₃:1448.4; Found: 1446.9.

Example 46 describes the synthesis of chirally controlled chimericoligonucleotide comprising both phosphate diester internucleotidiclinkage and modified internucleotidic linkages. The exemplaryoligonucleotides containing mixed morpholinoethyl phosphorothioatetriester/phosphodiester internucleotidic linkages were synthesized on anABI-394 DNA/RNA synthesizer according to the cycle summarized in TableE-13 using 1 μmol synthesis column and 1.5 μmol of oxalyl linked dT onHCP. As each iterative cycle allows for different sulfurization oroxidation reagents to be reacted, one thiosulfonate reagent was used forsulfurization in one cycle and iodine-promoted oxidation was used inanother cycle. The synthesis was performed with removal of the terminal5′-O-DMTr group (DMT Off). The solid support was washed with dry ACN anddried under a flux of argon. The dry HCP was placed in a plastic vialand was treated with 1 mL of dry propylamine in dry pyridine (in a 1:4ratio) for a period of 18 h at r.t. The solvents were then evaporatedand the residue was re-suspended with ˜pH 1.5 aqueous solutioncontaining 10% DMSO and the HCP support was filtered off. The crudeproduct was purified by reverse phase preparative HPLC (Gradient of 5 to65% ACN in 20 mM sodium phosphate buffer, pH=6.0). The fractions havingpurity above 95% were pooled, concentrated and desalted by reverse-phaseHPLC (Gradient of 0 to 80% ACN). The final desalted product waslyophilized from water.

TABLE E-13 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI-394 Used for the Synthesis of Example 46. delivery timestep reaction reagent (sec) wait time (sec) 1 detritylation 3% TCA inDCM 3 + 60 + 10 N.A. 2 coupling 0.15M phosphoramidite in 5 + 4 30 + 600ACN + 1.2M CMPT in ACN 3 capping 1 5% Pac₂O in THF/2,6-lutidine 20 60 4capping 2 5% Pac₂O in THF/2,6-lutidine + 20 60 16% NMI in THF 5sulfurization 0.3M S-Morpholinoethyl 10 + 4 × 2 300 + 3 × 150 +Toluylthiosulfonate 600 

6 oxidation 0.02M I₂, Pyridine/Water 10 300 

Example 46. Synthesis of Oligonucleotide 152 (All-(Sp)-d[Cs1AGs1T])

Oligonucleotide 152 was synthesized as described above. RT in RP-HPLC(HPLC method 2): 13.42 min. UPLC/ESI-MS: Calcd for C₅₁H₇₂N₁₇O₂₂P₃S₂:1432.3; Found: 1431.7.

Example 47 describes the synthesis of chirally controlled chimericoligonucleotide comprising a phosphodiester internucleotidic linkage andboth modified chirally pure phosphotriester and phosphodiesterinternucleotidic linkages. The chirally controlled oligonucleotidescontaining mixed morpholinoethyl phosphorothioatetriester/phosphodiester/phosphorothioate diester linkages weresynthesized on an ABI-394 DNA/RNA synthesizer according to the cyclesummarized in Table E-14 using 1 μmol synthesis column and 1.5 μmol ofoxalyl linked dT on HCP. As each iterative cycle allows for differentsulfurization or oxidation reagents to be reacted, two differentthiosulfonate reagents were used for the two different sulfurizationcycles and iodine-promoted oxidation was used for another cycle. Thesynthesis was performed with removal of the terminal 5′-O-DMTr group(DMT Off). The solid support was washed with dry ACN and dried under aflux of argon. The dry solid support was then treated with 5 mL ofanhydrous 1 M solution of 1,5-diazabicyclo(4.3.0)non-5-ene (DBN) in dryACN-trimethylsilyl chloride—16:1 (v/v) for 10 min at r.t. The DBNsolution was slowly pushed through the column by means of a plastic luersyringe fixed to the outlet of the column. The support was then washedwith dry ACN and dried under vacuum. The dry HCP was placed in a plasticvial and was treated with 1 mL of dry propylamine in dry pyridine (in a1:4 ratio) for a period of 18 h at r.t. The solvents were thenevaporated and the residue was re-suspended with ˜pH 1.5 aqueoussolution containing 10% DMSO and the HCP support was filtered off. Thecrude product was purified by reverse phase preparative HPLC (Gradientof 5 to 65% ACN in 20 mM sodium phosphate buffer, pH=6.0). The fractionshaving purity above 95% were pooled, concentrated and desalted byreverse-phase HPLC (Gradient of 0 to 80% ACN). The final desaltedproduct was lyophilized from water.

TABLE E-14 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI-394 Used for the Synthesis of Example 47. delivery timewait time step reaction reagent (sec) (sec) 1 detritylation 3% TCA inDCM 3 + 60 + 10 N.A. 2 coupling 0.15M phosphoramidite in 5 + 4 30 + 600ACN + 1.2M CMPT in ACN 3 capping 1 5% Pac₂O in THF/2,6-lutidine 20  60 4capping 2 5% Pac₂O in THF/2,6-lutidine + 20  60 16% NMI in THF 5sulfurization 0.3M S-cyanoethyl 10 + 4 × 2 300 + 3 × 150 + 1MethylThioSulfonate 600

6 sulfurization 0.3M S-Morpholinoethyl 10 + 4 × 2 300 + 3 × 150 + 2ToluylThioSulfonate 600

7 oxidation 0.02M I₂, Pyridine/Water 10 300

Example 47. Synthesis of Oligonucleotide 153 (All-(Sp)-d[CAs1GsT])

Oligonucleotide 153 was synthesized as described above. RT in RP-HPLC(HPLC method 2): 11.48 min. UPLC/ESI-MS: Calcd for C₄₅H₆₁N₁₆O₂₁P₃S₂:1318.1; Found: 1318.1.

As will be appreciated by a person skilled in the art, followingexamples 46 and 47, and other examples and methods described herein,longer chimeric chirally controlled oligonucleotides can be prepared.

Chirally Pure Oligonucleotides have Different Properties than theMixture of Diastereomers from Non-Stereospecific Synthesis

As described above and herein, in some embodiments, the presentapplication provides chirally pure oligonucleotides that have differentchemical and biological properties than the mixture of diastereomerswith the same base sequence but synthesized through non-stereospecificoligonucleotide synthesis.

Example 48. HPLC Profile of Chirally Pure Oligonucleotides and Mixtureof Diastereomers from Non-Stereospecific Synthesis

Chirally pure phosphorothioate diester oligonucleotides A (Full R_(P),Oligonucleotide 101) and B (Full S_(P), Oligonucleotide 102) andnon-stereospecific C (stereorandom phosphorothioate diesterinternucleotidic linkages), which was made by standardnon-stereospecific oligonucleotide synthesis, were analyzed by the sameRP-HPLC conditions and the corresponding HPLC traces are represented onFIG. 2.

As clearly demonstrated in FIG. 2, the stereochemistry of thephosphorothioate diester 20-mer oligonucleotides affects their behavioras determined by RP-HPLC and IEX-HPLC. Without the intention to belimited by theory, a correlation is observed between the retention times(RT) obtained by RP-HPLC and IEX-HPLC, as RT trends are conserved. Thefull R_(P) stereoisomer (A) has a shorter retention than the full S_(P)stereoisomer (B), while the stereorandom phosphorothioate diesteroligonucleotide (C), being a mixture of all 2¹⁹ stereoisomers, has abroad HPLC peak, eluting between the extreme full R_(P) and full S_(P)diastereoisomers.

As will be appreciated by those skilled in the art, the presented dataherewith confirm that depicted analyses comparing different chirallycontrolled or uncontrolled (e.g., stereorandom) oligonucleotidecompositions of the same sequence show that the exemplified chirallyuncontrolled oligonucleotide composition (i.e., as prepared bynon-chiral controlled oligonucleotide synthesis), includes only anextremely low level of certain oligonucleotide types, such as the fullRp or Sp type.

Example 49. Thermal Denaturation Experiment (Tm)

Each DNA strand was mixed with complementary RNA in equimolarconcentration of 0.5 μM in 1×PBS. Total 2.5 mL solution was prepared foreach duplex and the mixture was heated at 90° C. for 2 min and wasallowed to cool down over the course of several hours. The mixtures werethen stored at 4° C. for 2 hrs. Absorbance at 254 nm was recorded at aninterval of 0.5 min starting the temperature gradient from 15° C. to 90°C. with rise of 0.5° C./minute, using a Perkin ElmerUV-spectrophotometer equipped with a Peltier unit. The 254 nm absorbancewas plotted against the temperature and the Tm values were calculatedfrom the respective first derivative of each curve.

FIG. 3 illustrates the difference in Tm between two stereopurediastereoisomer phosphorothioate oligonucleotides (full R_(P) 20-mer Aand full S_(P) 20-mer B) and the stereorandom phosphorothioateoligonucleotide (C). Full R_(P) phosphorothioate DNA demonstrate higheraffinity towards complementary RNA when compared to both the oppositefull S_(P) diastereoisomer and the stereorandom 20-mer.

Table E-15 below summarizes the differences between chirally controlledoligonucleotides and stereorandom oligonucleotide.

TABLE E-15 Differences between chirally controlled and stereorandomoligonucleotides. RT-IEX IC₅₀ (nM) (rounded Oligonucleotide (min) Tm (°C.) ± 0.5 up to single digit) 101 14.70 68.3 4 102 15.49 60.5 6 10315.10 63.4 6 104 15.04 63.5 4 105 14.75 66.5 3 106 15.43 61.8 4 10715.02 64.5 4 108 15.10 64.8 3 109 14.91 65.8 2 110 15.24 62.9 7 11115.69 NA NA 113 15.72 NA NA 114 14.14 65.2 5 115 14.30 65.2 3 116 14.1764.8 2 118 15.04 64.5 3 119 6.90 73.4 Not detected 120 15.49 65.3 4

Example 50. Biological Activity of Chirally Pure Oligonucleotides

After checking OD all oligonucleotide candidate molecules were dilutedto a starting concentration of 20 μM. Multidose forward transfectionexperiment was setup in 96-well plates using Hep3B cells (ATCC®, Cat.HB-8064™) using Lipofectamine 2000 (Life Technologies, Cat. 11668-019)transfection reagent.

Transfection Protocol:

2×10⁴ Hep3B cells per well were seeded and incubated for 24 hours at 37°C. in a CO₂ incubator in 100 μl of antibiotic free MEM medium (LifeTechnologies, Cat. 11095098) containing 10% FBS and 1% Glutamax I (LifeTechnologies, Cat. 35050061). Twelve dilutions were setup in nucleasefree water (Table E-16).

TABLE E-16 Concentrations of oligonucleotide transfection stock plate.Dilution 1 2 3 4 5 6 In μM 20 10 3.33333 1.11111 0.37037 0.12346Dilution 7 8 9 10 11 12 In μM 0.04115 0.01372 0.00457 0.00152 0.000510.00017

For each well, 0.5 μL of Lipofectamine 2000 were mixed with 9.5 μL ofOpti MEM I reduced serum medium (Life Technologies, Cat. 31985062) andincubated for 5 minutes. Following incubation, 10 μL of each of thecandidates at the reported concentrations was mixed with the dilutedLipofectamine 2000 by gentle pipetting. The mixture was then incubatedfor 20 minutes at room temperature to allow for complex formation.During this time the cell growth medium was replaced with 80 μL of freshantibiotic free MEM as previously described. 20 μL of lipid-oligocomplex was gently mixed into each well bringing the total volume to 100μL. The cells were then incubated for 24 hours at 37° C. in a CO₂incubator.

RNA Extraction:

After 24 hour incubation the cell growth medium was removed and RNA wasextracted using a Dynabeads mRNA Direct kit (Life Technologies, Cat.61012) per the instructions provided in the kit manual withoutmodifications. This magnetic Oligo (dT)²⁵ bead system allows forcost-effective and robust high-throughput poly (A) RNA extractioncircumventing DNAse treatment. RNA was eluted in nuclease-free water andstored at −80° C.

cDNA Synthesis:

10 μL of RNA was used in a 20 μL cDNA synthesis reaction using a HighCapacity cDNA Reverse Transcription Kit (Life Technologies, Cat.4374967) using the kit protocol with RNAse Inhibitor. The reversetranscription was performed in a 96-well format on a C1000 Touch thermalcycler (Biorad, Cat. 185-1196EDU).

Gene Expression Analysis:

Gene expression was measured by quantitative PCR using a LightCycler®480 SYBR Green I Master (Roche, Cat. 04707516001) on LightCycler 480Real-Time PCR instrument. Primers for the Homo Sapiens sequences of thetarget Apolipoprotein B (Apo B) (NM_000384) and endogenous controlGlyceraldehyde-3-phosphate dehydrogenase (GAPDH)

TABLE E-17 Sequences of HPLC purified primersused for gene expression quantification. Primer SEQ ID Name TargetSequence NO: Position Apo B H. sapiens AGCCTTGGTG 116 3209-3232 ForwardApo B GATACCCTGA primer AGTT Apo B H. sapiens TGGACAAGGT 117 3289-3312Reverse Apo B CATACTCTGC primer CGAT GAPDH H. sapiens CTCTGCTCCT 11830-49 Forward GAPDH CCTGTTCGAC primer GAPDH H. sapiens ACGACCAAAT 119122-141 Reverse GAPDH CCGTTGACTC primer

Measurement of the target and endogenous control was performed inseparate wells using material from the same cDNA template. SYBR greenassay PCR conditions were setup as described in the SYBR Green I Masterprotocol (Table E-18).

TABLE E-18 Real-time PCR conditions for SYBR green assay. Step Number ofCycles Pre-incubation 1 Amplification 45 Melting curve 1 Cooling 1Acquisition Acquisition per Degrees ° C. Time Mode Ramp rate ° C./s ° C.Pre-incubation 95 5 min None 4.4 None Amplification 95 10 sec None 4.4None 60 10 sec None 2.2 None 72 10 sec Single 4.4 None Melting Curve 955 sec None 4.4 None 65 1 min None 2.2 None 97 Continuous 0.11 5 Cooling40 30 sec None 2.2 None

Melt Curve Analysis show single amplicon peaks for each primer pair(FIG. 4).

The data is a result of 3 biological replicates. All samples werecompared to Naïve untransfected control. The following results wereobserved when evaluating gene expression by real-time PCR using SYBRgreen (Table E-19, FIG. 5).

TABLE E-19 Complete IC₅₀ data evaluated by SYBR green. OligonucleotideIC₅₀ (nM) 101 4.368 All-R 102 6.345 All-S 103 5.727 5R-9S-5R 104 4.2915S-9R-5S 105 2.877 1S-17R-1S 106 ~3.855 1R-17S-1R 107 ~4.180 (R/S)₉R 1083.189 (S/R)₉S 118 3.087 Stereorandom 109 1.722 3S-13R-3S 110 7.4373R-13S-3R 114 5.234 3R-13S-3R 115 2.689 S-(RRS)₆ 116 1.919 RS-(RRS)₅-RR120 4.386 Mipomersen

In some embodiments, provided chirally controlled oligonucleotides showan IC₅₀ below 8 nM. In some embodiments, provided chirally controlledoligonucleotides show an IC₅₀ below 7 nM. In some embodiments, providedchirally controlled oligonucleotides show an IC₅₀ below 6 nM. In someembodiments, provided chirally controlled oligonucleotides show an IC₅₀below 5 nM. In some embodiments, provided chirally controlledoligonucleotides show an IC₅₀ below 4 nM. In some embodiments, providedchirally controlled oligonucleotides show an IC₅₀ below 3 nM. In someembodiments, provided chirally controlled oligonucleotides show an IC₅₀below 2 nM. In some embodiments, provided chirally controlledoligonucleotides show an IC₅₀ within a range of 0.5-8 nM. In someembodiments, provided chirally controlled oligonucleotides show an IC₅₀within a range of 1-4 nM. In some embodiments, provided chirallycontrolled oligonucleotides show an IC₅₀ within a range of 1.5-3 nM. Insome embodiments, provided chirally controlled oligonucleotides show anIC₅₀ within a range of 1.5-2 nM. In some embodiments, provided chirallycontrolled oligonucleotides show an IC₅₀ within a range of 0.5-2 nM. Insome embodiments, provided chirally controlled oligonucleotides show anIC₅₀ within a range of 1-2.5 nM. In some embodiments, provided chirallycontrolled oligonucleotides show an IC₅₀ within a range of 1.5-3 nM. Insome embodiments, provided chirally controlled oligonucleotides show anIC₅₀ within a range of 2.5-5 nM. In some embodiments, provided chirallycontrolled oligonucleotides show an IC₅₀ within a range of 3-6 nM. Insome embodiments, provided chirally controlled oligonucleotides show anIC₅₀ within a range of 5-8 nM. In some embodiments, provided chirallycontrolled oligonucleotides show an IC₅₀ within a range between an upperboundary and a lower boundary. In some such embodiments, the upperboundary is 8, 5, 4, or 3 nM. In some such embodiments, the lowerboundary is 1, 1.5, 2, or 2.5 nM. As can be seen with reference to theExamples, the present disclosure specifically exemplifies variouschirally controlled oligonucleotides or chirally controlledoligonucleotide compositions showing representative such IC₅₀ values.

Table E-19 illustrates that when test in chirally controlled form,certain chirally controlled oligonucleotides/oligonucleotidecompositions are more potent than the corresponding stereorandomoligonucleotide mixture (Oligonucleotides 105, 109, 115 and 116 comparedto Oligonucleotide 118); they are also more active than Mipomersen,which is a stereorandom oligonucleotide mixture (Oligonucleotides 104,105, 106, 107, 109, 115 and 116 compared to 120).

Additional Chirally Controlled Preparations Example 51. Preparation ofChirally Controlled Preparations of Oligonucleotides

The present Example describes preparation of a variety of particularchirally controlled composition of certain oligonucleotides as describedherein. In particular, the present Example describes oligonucleotidepreparation on utilizing loaded lcaa-CPG-500.

N⁴-Bz-5′-O-DMTr-2′-O-MOE-5mC (1) (4.0 g, 5.5 mmol) was dissolved inanhydrous DCM (20 mL) and mixed with 2 equiv of succinic anhydride (1.1g, 11.1 mmol) and 3 equiv of 4-N,N-dimethylaminopyridine (2.0 g, 16.6mmol). The reaction was stirred under Argon at room temperature. Aftercomplete consumption of the starting material as determined by TLC (1hour), the solvents were evaporated to dryness, the crude residue wasdissolved in DCM containing 1% triethylamine then purified by flashsilica gel chromatography using a gradient of 0-2% of MeOH in DCMcontaining 2% triethylamine. Yield of pure compound (2) afterevaporation was 4.5 g, 88%. The resulting 3′-O-succinate (2) (0.92 g,1.0 mmol), N,N-diisopropylethylamine (0.82 ml, 5.0 mmol) and CPG-500 (10g) were taken up in DMF (50 mL) then HBTU (0.42 g, 1.1 mmol) was added.The mixture was shaken for 2 h then filtered. The support was washedwith DMF, MeOH and finally, DCM then dried in vacuo. Trityl cationanalysis (monitoring at 504 nm) showed that the loading of thenucleoside on the support (3) was 63 μmol/g.

Chirally controlled compositions of oligonucleotides containingstereodefined chimeric deoxy and 2′-O-MOE phosphorothioate triesterinternucleotidic linkages was synthesized on MerMade-12 DNA/RNAsynthesizer (BioAutomation) according to the cycle summarized in TableE-20 using a MM-6-200 synthesis column (BioAutomation) loaded with 2.0 g(126 μmol) of uncapped succinyl linked N⁴-Bz-5′-O-DMTr-2′-O-MOE-5mC (63μmol/g, lcaa CPG-500 from Glen Research. The oligonucleotide synthesiscycle was performed with a preliminary capping step (capping 2) andwithout removal of the final terminal 5′-O-DMTr oligonucleotide group(DMT On). The stereospecific sulfurization steps were performed usingthe 0.3 M S-(2-cyanoethyl)methylthiosulfonate reagent following thecoupling of the corresponding chiral phosphoramide and the two-stepcapping process (Table E-20).

Once the automated oligonucleotide synthesis cycle was completed withthe final 5′-O-DMTr group kept On, the synthesis column was taken offthe DNA/RNA synthesizer and dried under vacuum. The dried support wastransferred onto an empty glass manual peptide synthesizer and 40 mLsolution of 0.5 M 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 0.25 MN,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in ACN wascontinuously passed through the support for 5 min without stopping theflow in the manual peptide synthesizer. The support was washed by 50%Py/ACN and dried with Argon flow for 1 sec. Then, the support wastransferred into an empty screw-cap plastic vial and treated with 5%EtOH/conc NH₃ (20 mL) at 60° C. for 6 h, and left at room temperaturefor 12 h. The support was removed by filteration and washed with conc.NH₃. The filtrate was dried in vacuo then the resultant was dissolved in50 mM TEAA (120 mL). Further filtration was performed when there was asuspension in the solution. The solution was loaded on a Sep-Pakcartridge (Waters, Sep-Pak Vac 35 cc (10 g) Cis Cartridges). Thecartridge was washed with 20% ACN/50 mM TEAA (70 mL) to remove allcapped truncated sequences and 50% ACN/water containing 0.5% of conc NH₃(50 mL) to elute the full length DMT On oligonucleotide. The solutioncontaining the DMT On oligonucleotide was dried in vacuo then dilutedwith 50 mM TEAA (120 mL) and loaded on another Sep-Pak cartridge(Waters, Sep-Pak Vac 35 cc (10 g) C₁₈ Cartridges). The cartridge waswashed with milli Q water (50 mL), 2% TFA/water (50 mL), then water (50mL). The DMT Off oligonucleotide was eluted with 50% ACN/watercontaining 0.5% of cone NH₃ (50 mL).

TABLE E-20 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer MerMade 12 Used for Chirally Controlled Synthesis. deliverywait volume time step reaction reagent (mL) (sec) 1 detritylation 3% TCAin DCM 3 × 10  3 × 24  2 coupling 0.15M chiral 2 × 3.4 2 × 450phosphoramidite in ACN + (MOE) 1.2M CMPT in ACN 2 × 300 (DNA) 3 capping1 5% Pac₂O in THF/2,6- 8 60 lutidine 4 capping 2 5% Pac₂O in THF/2,6-  6.8 60 lutidine + 16% NMI in THF 5 sulfurization 0.3M S-(2-cyanoethyl)9 600  methanethiosulfonate

Synthesis of Oligonucleotide ONT-75 (All (Rp))-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ ID NO: 106)

Synthesis of Oligonucleotide ONT-80 (All (Sp))-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ ID NO: 106)

Synthesis of Oligonucleotide ONT-77 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp,Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ ID NO: 106) (5R-10S-4R)

Synthesis of Oligonucleotide ONT-81 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ ID NO: 106) (5S-10R-4S)

Synthesis of Oligonucleotide ONT-87 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp,Sp, Rp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ ID NO: 106) (5R-(SSR)3-5R)

Synthesis of Oligonucleotide ONT-88 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp,Rp, Sp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ ID NO: 106) (5S-(RRS)3-5S)

Synthesis of Oligonucleotide ONT-89 (Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ ID NO: 106) ((SR)₉S)

Synthesis of Oligonucleotide ONT-82 (All(Rp))-GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC (SEQ ID NO: 120)

Synthesis of Oligonucleotide ONT-84 (All(Sp))-GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC (SEQ ID NO: 120)

Synthesis of Oligonucleotide ONT-85 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp,Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp,Rp)-GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC (SEQ ID NO: 120)(5R-10S-4R)

Synthesis of Oligonucleotide ONT-86 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp,Sp)-GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC (SEQ ID NO: 120)(5S-10R-4S)

Example 52: General RP-HPLC Method for the Analysis of Crude DMT On andPurified DMT Off Oligonucleotides

The present Example describes RP-HPLC analysis of crude and purifiedoligonucleotide compositions prepared by chirally controlled synthesisas described herein.

Buffer A: 50 mM TEAA, pH 7.0 Buffer B: ACN

Column: XBridge C₁₈, 3.5 μm, C₁₈, 4.6×50 mm, Part #186003034Column temperature=50° C.Signal monitored at 254 and 280 nmGradient used:

Time(min) Flow (mL/min) % A % B Curve Initial 99 1 2 1 99 1 6 52 1 50 506 55 1 5 95 6 55.5 1 5 95 6 56 1 99 1 6 60 1 99 1 1

SEQ. ID FIG. RP-HPLC of Crude DMT on: NO: 35: ONT-75 (All(Rp))-Gs5mCs5mCsTs5mCsAs 106 (Panel A)GsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC ONT-80 (All(Sp))-Gs5mCs5mCsTs5mCsAs 106 (Panel B)GsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC ONT-77: (Rp, Rp, Rp, Rp, Rp,Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, 106 (Panel C) Rp,Rp)-Gs5mCs5mCsTs5mCsAs GsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC(5R-10S-4R) ONT-81 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp,Rp, Rp, Sp, Sp, 106 (Panel D) Sp,Sp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC (5S-10R-4S) ONT-87 (Rp, Rp, Rp,Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Rp, Rp, Rp, 106 (Panel E)Rp,Rp)-Gs5mCs5mCsTs5mCsAs GsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC(5R-(SSR)₃-5R) ONT-88 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp,Rp, Sp, Sp, Sp, Sp, Sp, 106 (Panel F) Sp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC (5S-(RRS)₃-5S) ONT-89 (Sp, Rp,Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, 106 (PanelG) Rp,Sp)-Gs5mCs5mCsTs5mCsAs GsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC((SR)₉S) ONT-82 (All (Rp))- 120 (Panel H)GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC ONT-84 (All (Sp))- 120(Panel I) GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC ONT-85 (Rp,Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, 120(Panel J) Rp,Rp)-GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC(5R-10S-4R) ONT-86 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp,Rp, Rp, Sp, Sp, 120 (Panel K)Sp,Sp)-GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC (5S- 10R-4S)

Example 53: General IEX-HPLC Method for the Analysis of Crude DMT On andPurified DMT Off Oligonucleotides

The present Example describes IEX-HPLC analysis of crude and purifiedoligonucleotide compositions prepared by chirally controlled synthesisas described herein.

Buffer A: 10 mM TrisHCl, 50% ACN, pH 8.0 Buffer B: 10 mM TrisHCl, 800 mMNaClO₄, 50% ACN, pH 8.0 Column: DIONEX, DNAPac, PA-100, Analytical,4.0×250 mm, Part #063000

Column temperature=60° C.Signal monitored at 254 and 280 nmGradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 85 15 3 1 85 15 1 23 1 6040 6 25 1 5 95 6 25.5 1 85 15 6 30 1 85 15 1

Example 54: General UPLC-LCMS Method for the Analysis of Purified DMTOff Oligonucleotides

The present Example describes UPLC-LCMS analysis of purifiedoligonucleotide compositions prepared by chirally controlled synthesisas described herein.

Buffer A: 15 mM TEA, 400 mM HFIP, Water Buffer B: 50:50 BufferA/Methanol Column: UPLC@OST C₁₈ 1.7 μm, 2.1×500 mm

Column temperature=50° C.Gradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 0.2 80 20 2 0.2 80 20 122 0.2 30 70 6 25 0.2 5 95 6 25.5 0.2 80 20 6 30 0.2 80 20 1

Example 55: General IEX-HPLC Method for the Purification of Crude DMTOff Oligonucleotide

The present Example describes IEX-HPLC purification of crudeoligonucleotide compositions prepared by chirally controlled synthesisas described herein.

Buffer A: 20 mM NaOH, pH 11.0 Buffer B: 20 mM NaOH, 2.5 M NaCl, pH 11.0

Column: Empty column Waters AP-2 (Waters), custom in-house packed withSource 15Q support (GE Healthcare). Individual purification columns werepacked and used for the different stereopure oligonucleotides.Instrument: AKTA Purifier, equipped with the P-900 pump, the UPC-900detector and the 50 mL injection SuperLoop (GE Healthcare)Buffer heater temperature set=70° C.Signal monitored at 254 nmFractions volume: 5 mLGradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 100 0 25 10 100 0 1 40 1085 15 6 60 10 85 15 1 80 10 70 30 6 100 10 70 30 1 140 10 60 40 6 180 1060 40 1 200 10 45 55 6 210 10 45 55 1 211 10 100 0 6 235 10 100 0 1

Collected fractions were individually analyzed by analytical IEX-HPLCusing the analytical conditions and gradient described above. Purefractions were pooled in order to provide purified material of 95% andabove purity as determined by the 254 nm UV absorbance profiles.

SEQ ID FIG. RP-HPLC of Purified DMT Off NO: 36: ONT-75 (All(Rp))-Gs5mCs5mCsTs5mCsAs 106 (Panel A)GsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC ONT-80 (All(Sp))-Gs5mCs5mCsTs5mCsAs 106 (Panel B)GsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC ONT-77 (Rp, Rp, Rp, Rp, Rp, Sp,Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, 106 (Panel C)Rp)-Gs5mCs5mCsTs5mCsAs GsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC(5R-10S-4R) ONT-81 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp,Rp, Rp, Sp, Sp, Sp, 106 (Panel D) Sp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC (5S-10R-4S) ONT-87 (Rp, Rp, Rp,Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, 106 (PanelE) Rp)-Gs5mCs5mCsTs5mCsAs GsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC(5R-(SSR)₃-5R) ONT-88 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp,Rp, Sp, Sp, Sp, Sp, Sp, 106 (Panel F) Sp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC (5S-(RRS)₃-5S) ONT-89 (Sp, Rp,Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, 106(Panel G) Sp)-Gs5mCs5mCsTs5mCsAs GsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC ((SR)₉S) ONT-82 (All (Rp))- 120 (Panel H)GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC ONT-84 (All (Sp))- 120(Panel I) GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC ONT-85 (Rp,Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, 120(Panel J) Rp)-GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC (5R-10S-4R) ONT-86 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp,Rp, Sp, Sp, Sp, 120 (Panel K)Sp)-GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC (5S- 10R-4S)

Table E-21. Compilation of the Physico-Chemical Properties for theStereopure Phosphorothioate Oligonucleotides Synthesized as Described inExample XX.

Sequence A: human ApoB sequence5′-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC-3′ (SEQ IDNO: 106).

Sequence B: mouse ApoB sequence5′-GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC-3′ (SEQ ID NO:120).

Underlined nucleotides designate 2′-O-MOE. s=phosphorothioate linkage.5mC=5-methyl-2′-deoxycytidine. 5mC=5-methyl-2′-O-MOE-cytidine. Stereoarchitecture describes the stereoisomer nature (Rp/Sp) of eachphosphorus atom in a given phosphorothioate linkage of theoligonucleotide. Retention time (t_(R)) in IEX-HPLC and Found molecularweight (MW) values were obtained using the corresponding analyticalmethods for the purified compounds (described above).

t_(R) IEX- sequence HPLC Calc Found ID sequence stereo architecture(min) MW MW ONT-41 A Stereorandom 17.34 7177.2 7175.7 diastereomixtureONT-75 A All Rp 16.44 7177.2 7177.0 ONT-77 A 5R-10S-4R 17.44 7177.27177.5 ONT-80 A All Sp 18.13 7177.2 7175.5 ONT-81 A 5S-10R-4S 17.677177.2 7175.8 ONT-87 A 5R-(SSR)₃-5R 13.64 7177.2 — ONT-88 A 5S-(RRS)₃-5S14.88 7177.2 7177.0 ONT-89 A (SR)₉S 14.25 7177.2 — ONT-83 B Stereorandom17.02 7233.2 7231.8 diastereomixture ONT-82 B All Rp 16.43 7233.2 7231.6ONT-84 B All Sp 19.51 7233.2 7232.1 ONT-85 B 5R-10S-4R 18.76 7233.27231.1 ONT-86 B 5S-10R-4S 18.75 7233.2 7230.9

Example 56: General RP-HPLC Method for the Overlay Analyses of PurifiedDMT Off Oligonucleotides

The present Example describes RP-HPLC analysis of purifiedoligonucleotide compositions prepared by chirally controlled synthesisas described herein.

Buffer A: 50 mM TEAA, pH 7.0 Buffer B: ACN

Column: XBridge C₁₈, 3.5 μm, C₁₈, 4.6×50 mmColumn temperature=50° C.Signal monitored at 254 and 280 nmGradient used:

Time(min) Flow (mL/min) % A % B Curve Initial 95 5 2 1 95 5 1 52 1 70 306 54 1 5 95 6 54.5 1 5 95 1 55 1 99 1 6 60 1 99 1 1

Panel A of FIG. 37 is an overlay of RP-HPLC traces of Purified DMT OffOligonucleotide ONT-75, ONT-77, ONT-80, ONT-81, ONT-87, ONT-88, ONT-89and ONT-41(diastereomixture)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC (SEQ ID NO: 106).

Panel B of FIG. 37 shows an expanded view of Panel A, with each curvelabeled as follows:

Curve # Oligonucleotide 1 diastereomixture (ONT-41) 2 all-Rp (ONT-75) 35R-(SSR)₃-5R (ONT-87) 4 (SR)₉-S (ONT-89) 5 5R-10S-4R (ONT-77) 65S-10R-4S (ONT-81) 7 5S-(RRS)₃-5S (ONT-88) 8 all-Sp (ONT-80)

Panel A of FIG. 38 is an overlay of RP-HPLC of Purified DMT OffOligonucleotide ONT-82, ONT-84, ONT-85, ONT-86, and ONT-83(diastereomixture)-GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC(SEQ ID NO: 120)

Panel B of FIG. 38 shows an expanded view of Panel A, with each curvelabeled as follows:

Curve # Oligonucleotide 1 diastereomixture (ONT-83) 2 all-Rp (ONT-82) 35S-10R-4S (ONT-86) 4 5R-10S-4R (ONT-85) 5 all-Sp (ONT-84)

Example 57: Thermal Denaturation Experiment (Tm)

The present Example describes characterization of chirally controlledoligonucleotide compositions using thermal denaturation.

Each DNA strand was mixed with its complementary RNA strand in equimolarconcentration of 1 μM in 1×PBS. Total 3 mL solution was prepared foreach duplex and the mixture was heated at 90° C. for 2 min and wasallowed to cool down over the course of several hours. The mixtures werethen stored at 4° C. for 2 hrs. Absorbance at 254 nm was recorded at aninterval of 0.5 min starting the temperature gradient from 15.0° C. to95.0° C. with rise of 0.5° C./minute, using Cary100 Series (AgilentTechnologies). The 254 nm absorbance was plotted against the temperatureand the Tm values were calculated from the respective first derivativeof each curve.

FIG. 39 presents a Tm overlay of chirally controlled oligonucleotidesand stereorandom oligonucleotide.

FIG. 39 illustrates the difference in Tm between four stereopurediastereoisomer phosphorothioate oligonucleotides (all-Rp 20-mer, all-Sp20-mer, 5R-10S-4R and 5S-10R-4S gapmers) and the stereorandomphosphorothioate oligonucleotide. Full Rp phosphorothioate DNAdemonstrates the highest affinity towards complementary RNA (Tm=85.1°C.) when compared to full Sp 20-mer (Tm=75.1° C.) which has the lowestaffinity. 5R-10S-4R and 5S-10R-4S gapmers and the stereorandomphosphorothioate oligonucleotides all showed intermediates values (Tmrange=80.1-81.2° C.).

Table E-22, below, gives Tm values for the studied stereopure andstereorandom phosphorothioate oligonucleotides having the human ApoBsequence 5′-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC-3′(SEQ ID NO: 106) with various stereochemistry architectures on thephosphorothioate backbone.

TABLE E-22 sequence ID stereo architecture Tm (° C.) ONT-41 Stereorandom81.0 diastereomixture ONT-75 All Rp 85.1 ONT-77 5R-10S-4R 80.1 ONT-80All Sp 75.1 ONT-81 5S-10R-4S 81.2

Example 58: Preparation of Exemplary Chirally Controlled siRNAOligonucleotides Targeting PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9), is an enzymeinvolved in cholesterol metabolism. PCSK9 binds to the receptor for lowdensity lipoprotein (LDL), triggering its destruction. Although LDLassociated with the receptor is also eliminated when the receptor isdestroyed, the net effect of PCSK9 binding in fact increases LDL levels,as the receptor would otherwise cycle back to the cell surface andremove more cholesterol.

Several companies are developing therapeutic agents that target PCSK9.Of particular relevance to the present disclosure, each of IsisPharmaceuticals, Santaris Pharma, and Alnylam Pharmaceuticals isdeveloping a nucleic acid agent that inhibits PCSK9. The IsisPharmaceuticals product, an antisense oligonucleotide, has been shown toincrease expression of the LDLR and decrease circulating totalcholesterol levels in mice (Graham et al “Antisense inhibition ofproprotein convertase subtilisin/kexin type 9 reduces serum LDL inhyperlipidemic mice”. J. Lipid Res. 48 (4): 763-7, April 2007). Initialclinical trials with the Alnylam Pharmaceuticals product, ALN-PCS,reveal that RNA interference offers an effective mechanism forinhibiting PCSK9 (Frank-Kamenetsky et al “Therapeutic RNAi targetingPCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterolin nonhuman primates”. Proc. Natl. Acad. Sci. U.S.A. 105 (33): 11915-20,August 2008).

The present Example describes preparation of a variety of chirallycontrolled or stereorandom siRNA agents directed to PCSK9. Specifically,this Example describes preparation of each of the followingoligonucleotide compositions as illustrated in Table E-23 below:

TABLE E-23 SEQ ID ONT # NO: Sequence Type ONT-106 121 (Rp)-uucuAGAccuGuPCSK9 uuuGcuudT

dT sense ONT-107 121 (Sp)-uucuAGAccuGu PCSK9 uuuGcuudT

dT sense ONT-108 122 (Rp)-AAGcAAAAcAGG PCSK9 UCuAGAAdTsdt antisenseONT-109 122 (Sp)-AAGcAAAAcAGG PCSK9 UCuAGAAdT

dT antisense ONT-110 122 (Rp, Rp)-asAGcAAA PCSK9 AcAGGUCuAGAAdT

dT antisense ONT-111 123 (Sp, Rp)-asGcAAAA PCSK9 cAGGUCuAGAAdT

dT antisense ONT-112 123 (Sp, Sp)-asGcAAAA PCSK9 cAGGUCuAGAAdT

dT antisense ONT-113 123 (Rp, Sp)-asGcAAAA PCSK9 cAGGUCuAGAAdT

dT antisense NOTE: lower case letters represent 2′OMe RNA residues;capital letters represent 2′OH RNA residues; and bolded and italicized“s” indicates a phosphorothioate moiety.

Lcaa-CPG-500 used in the preparation of these oligonucleotides wasprepared according to the following reaction:

In particular, 5′-O-DMTr-2′-deoxythymidine (1) (4.28 g, 7.86 mmol) wasdissolved in anhydrous DCM (50 mL) and mixed with 2 equiv of succinicanhydride (1.57 g, 15.7 mmol) and 3 equiv of 4-N,N-dimethylaminopyridine(2.88 g, 23.6 mmol). The reaction was stirred under Argon at roomtemperature. After complete consumption of the starting material asdetermined by TLC (1 hour), the solvents were evaporated to dryness, thecrude residue was dissolved in DCM containing 1% triethylamine thenpurified by flash silica gel chromatography using a gradient of 0-2% ofMeOH in DCM containing 2% triethylamine. Yield of pure compound (2)after evaporation was 5.5 g, 94%. The resulting5′-O-DMTr-2′-deoxythymidine-3′-O-succinate (2) (0.60 g, 0.81 mmol),N,N-diisopropylethylamine (0.71 mL, 4.02 mmol) and CPG-500 (10 g) weretaken up in DMF (50 mL) then HBTU (0.37 g, 0.97 mmol) was added. Themixture was shaken for 2 h then filtered. The support was washed withDMF, MeOH and finally, DCM then dried in vacuo. Trityl cation analysis(monitoring at 504 nm) showed that the loading of nucleoside on thesupport (3) was 38 μmol/g.

Each oligonucleotide, containing 2′-OH and 2′-OMe phosphodiester andstereodefined 2′-deoxy and 2′-OMe phosphorothioate diesterinternucleotidic linkages as indicated above in Table E-23, wassynthesized on an ABI 394 DNA/RNA synthesizer according to the cyclessummarized in Table E-24 and Table E-25 respectively, using a 10 μmolcapacity synthesis column loaded with 130 mg (4.9 μmol) of succinyllinked 5′-O-DMTr-2′-deoxythymidine (38 μmol/g). Prior to synthesis, apreliminary capping step (capping 2) was performed and the synthesis wasterminated with removal of the terminal 5′-O-DMTr groups. The oxidationstep was performed using a commercially available 5-6 M solution oftert-butyl hydroperoxide (TBHP) in decane which was then diluted withfour parts dichloromethane. The stereospecific sulfurization step wasperformed using the 0.3 M S-cyanoethylmethylthiosulfonate reagentfollowing the coupling of the corresponding chiral phosphoramide and thetwo-step capping process (Table E-25).

Once the automated oligonucleotide synthesis cycle was completed and thefinal 5′-O-DMTr group was removed, the synthesis column was taken offthe DNA/RNA synthesizer and dried under vacuum. 10 mL solution of 0.5 M1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 0.25 MN,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in ACN wascontinuously added to the support through the synthesis column for 1 minwithout stopping the flow using a syringe attached to one end of thesynthesis column. The support was then washed with anhydrous ACN anddried under vacuum. Then, the dried support was transferred into anempty screw-cap plastic vial and treated with 40% MeNH₂ aqueous solution(0.5 mL) at 60° C. for 10 min. After that, the vial was immediatelycooled down and after dilution with DMSO (0.5 mL), the support wasremoved by filtration, washed again with DMSO (1 mL) and filtered. Thefiltrate was cooled down and then immediately treated with 3HF.Et₃N(0.75 mL) at 60° C. for 10 min, then immediately frozen and stored at 4°C. prior to purification.

TABLE E-24 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI 394 (2′-O-TBDMS and 2′-OMe substituted RNA cycle) stepreaction reagent delivery time (sec) wait time (sec) 1 detritylation 3%TCA in DCM 3 + 120 + 10 N. A. 2 coupling 0.15M 7 + 6 30 + 600phosphoramidite in ACN + 2M CMPT in ACN 3 capping 5% Pac₂O in THF/2,6-10 20 lutidine + 16% NMI in THF 4 oxidation 1.1M tert-butyl 20 110hydroperoxide in 4:1 dichloromethane:decane

TABLE E-25 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI 394 (stereodefined phosphorothioate 2′-deoxy and 2′-OMeRNA cycle) step reaction reagent delivery time (sec) wait time (sec) 1detritylation 3% TCA in DCM 3 + 120 + 10 N.A. 2 coupling 0.15M chiral8 + 6 30 + 900 (2′-OMe phosphoramidite in RNA) ACN + 2M CMPT in 30 + 600(DNA) ACN 3 capping 1 5% Pac₂O in THF/2,6- 30 60 lutidine 4 capping 2 5%Pac₂O in THF/2,6- 30 60 lutidine + 16% NMI in THF 5 sulfurization 0.3MS-(2-cyanoethyl) 15 + 3 × 4 120 + 3 × 60 + 300 methylthiosulfonate

Example 59. General IEX-HPLC Method for the Analysis of Crude andPurified DMT Off RNA Oligonucleotides

Buffer A: 20 mM sodium phosphate, pH 11.0Buffer B: 20 mM sodium phosphate, 1 M NaBr, pH 11.0

Column: DIONEX, DNAPac, PA-100, Analytical, 4.0×250 mm

Column temperature=60° C.Signal monitored at 254 and 280 nmGradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 95 5 3 1 95 5 1 23 1 2080 6 25 1 5 95 6 25.5 1 95 5 6 30 1 95 5 1

Example 60. General UPLC-LCMS Method for the Analysis of Purified DMTOff RNA Oligonucleotides Buffer A: 15 mM TEA, 400 mM HFIP, Water BufferB: 50:50 Buffer A/Methanol Column: UPLC@OST C₁₈ 1.7 μm, 2.1×500 mm

Column temperature=50° C.Gradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 0.2 95 5 10 0.2 35 65 612 0.2 5 95 6 12.5 0.2 95 5 6 15 0.2 95 5 1

Example 61. General IEX-HPLC Method for the Purification of Crude DMTrOff RNA Oligonucleotides

Buffer A: 20 mM sodium phosphate, pH 8.5Buffer B: 20 mM sodium phosphate, 1 M NaBr, pH 8.5Column: Empty column Waters AP-2 (Waters), custom in-house packed withSource 15Q support (GE Healthcare). The same purification column wasused for the different stereopure oligonucleotides.Instrument: Waters HPLC unit equipped with the 2525 binary gradientmodule, the 2487 dual absorbance detector and the 20 mL Flex injector(Waters).Buffer heater temperature set=70° C.Signal monitored at 254 nm and 280 nmFractions volume: 4.5 mLGradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 100 0 10 10 100 0 1 25 1080 20 6 85 10 67.5 32.5 6 95 10 67.5 32.5 1 155 10 55 45 6 165 10 0 1006 170 10 100 0 6 180 10 100 0 1

Collected fractions were individually analyzed by analytical IEX-HPLCusing the analytical conditions and gradient described above. Purefractions were pooled in order to provide purified material of 95% andabove purity as determined by the 254 nm UV absorbance profiles.

Example 62. General RP-Sep-Pak Method for the Desalting of Purified RNAOligonucleotides

The solution of pooled pure fractions was loaded on a Sep-Pak cartridge(Waters, Sep-Pak Vac 35 cc (10 g) C₁₈ Cartridges) pre-conditioned withwater. After loading of the sample (100 mL), the cartridge was washedwith milli Q water (50 mL) to remove all salt and then washed with 50%ACN/water to elute the full length desalted RNA oligonucleotide. Thecollected solution was concentrated in vacuo to a volume of 5 mL andlyophilized from water.

Example 63: Preparation of Double Stranded siRNA Agents Using ChirallyControlled Oligonucleotide Strands

The present Example describes preparation of double stranded siRNAagents by thermal annealing of chirally controlled oligonucleotidestrands as described above.

Each RNA strand was mixed with its complementary RNA strand in equimolarconcentration of 10 μM in 1×PBS. Total 0.5 mL solution was prepared foreach duplex and the mixture was heated at 90° C. for 2 min and wasallowed to cool down over the course of several hours. The mixtures werethen stored at 4° C. Physico-chemical properties of the utilized RNAstrands are presented below in Table E-26.

TABLE E-26 duplex sequence stereo Calc Found ID sequence ID architectureMW MW ONT D-1 S ONT-116 Stereorandom 6735.4 6734.9 diastereomixture AS1ONT-114 Stereorandom 6805.4 6805.2 diastereomixture ONT D-2 S ONT-107 Sp6735.4 6736.6 AS1 ONT-109 Sp 6805.4 6801.6 ONT D-3 S ONT-106 Rp 6735.46731.8 AS1 ONT-109 Sp 6805.4 6801.6 ONT D-4 S ONT-107 Sp 6735.4 6736.6AS1 ONT-108 Rp 6805.4 6799.2 ONT D-5 S ONT-106 Rp 6735.4 6731.8 AS1ONT-108 Rp 6805.4 6799.2 ONT D-7 S ONT-116 Stereorandom 6735.4 6734.9diastereomixture AS2 ONT-115 Stereorandom 6835.5 6835.2 diastereomixtureONT D-8 S ONT-106 Rp 6735.4 6731.8 AS2 ONT-110 Rp, Rp 6835.5 6832.3 ONTD-9 S ONT-107 Sp 6735.4 6736.6 AS2 ONT-110 Rp, Rp 6835.5 6832.2 ONT D-10S ONT-106 Rp 6735.4 6731.8 AS2 ONT-111 Sp, Rp 6835.5 6832.4 ONT D-11 SONT-107 Sp 6735.4 6736.6 AS2 ONT-111 Sp, Rp 6835.5 6832.4 ONT D-12 SONT-106 Rp 6735.4 6731.8 AS2 ONT-112 Sp, Sp 6835.5 6836.2 ONT D-13 SONT-107 Sp 6735.4 6736.6 AS2 ONT-112 Sp, Sp 6835.5 6836.2 ONT D-14 SONT-106 Rp 6735.4 6731.8 AS2 ONT-113 Rp, Sp 6835.5 6834.6 ONT D-15 SONT-107 Sp 6735.4 6736.6 AS2 ONT-113 Rp, Sp 6835.5 6834.6

Oligonucleotide sequences used, human PCSK9 siRNA: Sense strand (S)5′-uucuAGAccuGuuuuGcuudTsdT-3′ (SEQ ID NO: 121); Antisense strand 1(AS1) 5′-AAGcAAAAcAGGUCuAGAAdTsdT-3′ (SEQ ID NO: 122); Antisense strand2 (AS2) 5′-asAGcAAAAcAGGUCuAGAAdTsdT-3′ (SEQ ID NO: 123). Upper casenucleotides: RNA, lower case nucleotides 2′-OMe, d=2′-deoxy,s=phosphorothioate. Stereo architecture describes the stereoisomernature (Rp/Sp absolute configuration) of each phosphorus atom in a givenphosphorothioate linkage of the oligonucleotide. Found molecular weight(MW) values were obtained using the corresponding analytical methods forthe purified compounds (described above).

FIG. 51 shows overlays of IEX-HPLC profiles showing difference inretention times between stereopure RNA oligonucleotides.

Curve # Oligonucleotide 1 ONT-109 (Sp) 2 ONT-114 (diastereomixture) 3ONT-108 (Rp)

FIG. 52 shows overlays of IEX-HPLC profiles showing difference inretention times between stereopure RNA oligonucleotides.

Curve # Oligonucleotide 1 ONT-109 (Rp) 2 ONT-114 (diastereomixture) 3ONT-107 (Sp)

In addition to the above specifically exemplified, prepared chirallycontrolled siRNA oligonucleotides, the present invention provides forpreparation of siRNA duplexes having several chiral phosphorothioateinternucleotide linkages, and full chiral phosphorothioateinternucleotide linkages.

For example, in accordance with the present invention, multiple chiralphosphorothioate linkages are introduced inside a RNA oligonucleotide byusing the appropriate chiral RNA 3′-phosphoramidites, having suitable2′-OH protecting groups, such as 2′-O-PivOM (Debart et al., Chem. Eur.J., 2008, 14, 9135), 2′-O-CEM (Ohgi et al., Org. Lett., 2005, 7, 7913;Wada et al., J. Org. Chem., 2012, 77, 7913), 2′-O-TOM (Pitsch et al.,Helv. Chim. Acta, 2001, 84, 3773) or 2′-O-TC (Dellinger et al., J. Am.Chem. Soc., 2011, 133, 11540). Each of the following Human PCSK9 siRNASense Strands having several chiral phosphorothioate internucleotidelinkages and full chiral phosphorothioate internucleotide linkages canbe prepared in accordance with the present invention:

SEQ ID NO: PCSK9 siRNA Sense Strands PCSK9 121 (All (Sp))- (1)ususcsusAsGsAscscsusGsususususGscsususdTsdT PCSK9 121 (All (Rp))- (2)ususcsusAsGsAscscsusGsususususGscsususdTsdT PCSK9 121 (All(Sp))-usucuAsGsAsccuGsuuuuGscuusdTsdT (3) PCSK9 121 (All(Rp))-usucuAsGsAsccuGsuuuuGscuusdTsdT (4) PCSK9 121 (Rp, Sp, Rp, Sp, Rp,Sp, Rp, Sp, Rp, Sp, Rp, Sp, (5) Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp)-ususcsusAsGsAscscsusGsususususGscsususdTsdT PCSK9 121 (Sp, Rp, Sp, Rp,Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, (6) Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp)-ususcsusAsGsAscscsusGsususususGscsususdTsdT NOTE: lower case lettersrepresent 2′-OMe RNA residues; capital letters represent RNA residues; d= 2′-deoxy residues; and “s” indicates a phosphorothioate moiety.

Synthesis examples for Human PCSK9 siRNA Antisense Strands havingseveral chiral phosphorothioate internucleotide linkages and full chiralphosphorothioate internucleotide linkages.

SEQ ID NO: Human PCSK9 siRNA Antisense Strands PCSK9 (7) 122 (All (Rp))-AsAsGscsAsAsAsAscsAsGsGsUsCsusAsGsAsAsdTsdT PCSK9 (8) 122 (All (Sp))-AsAsGscsAsAsAsAscsAsGsGsUsCsusAsGsAsAsdTsdT PCSK9 (9) 122 (All (Rp))-AsAGcAAAAcsAsGsGsUsCsusAsGsAsAsdTsdT PCSK9 (10) 122 (All (Sp))-AsAGcAAAAcsAsGsGsUsCsusAsGsAsAsdTsdT PCSK9 (11) 122 (All (Rp))-AAsGscsAsAsAsAscAGGUCuAGAAdTsdT PCSK9 (12) 122 (All (Sp))-AAsGscsAsAsAsAscAGGUCuAGAAdTsdT PCSK9 (13) 122 (All (Rp))-AsAsGscAsAsAsAscAsGsGsUsCsuAsGsAsAsdTsdT PCSK9 (14) 122 (All (Sp))-AsAsGscAsAsAsAscAsGsGsUsCsuAsGsAsAsdTsdT PCSK9 (15) 122 (All (Rp))-AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT PCSK9 (16) 122 (All (Sp))-AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT PCSK9 (17) 122 (Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp)-AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT PCSK9 (18) 122 (Sp, Rp, Sp, Rp,Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp)-AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT NOTE: lower case letters represent2′-OMe RNA residues; capital letters represent RNA residues; d =2′-deoxy residues; and “s” indicates a phosphorothioate moiety.

Alternatively or additionally, the present invention provides forpreparation of siRNA duplexes having several chiral phosphorothioateinternucleotide linkages and full chiral phosphorothioateinternucleotide linkages and fully modified ribose moieties.

For example, in certain embodiments, multiple chiral phosphorothioatelinkages are introduced inside a fully ribose-modified RNAoligonucleotide by using the appropriate chiral RNA 3′-phosphoramidites,having the corresponding desired ribose 2′-chemical modification, suchas 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl (2′-OMe). To give but a fewexamples, each of the following Human PCSK9 siRNA fully modified2′-F/2′-OMe Sense Strands having several chiral phosphorothioateinternucleotide linkages and full chiral phosphorothioateinternucleotide linkages, can be prepared.

SEQ ID NO: Human PCSK9 siRNA fully modified 2′-F/2′-OMe Sense StrandsPCSK9 (19) 121 (All (Rp))-UfsusCfsusAfsgsAfscsCfsusGfsusUfsusUfsgsCfsusUfsdTsdT PCSK9 (20) 121(All (Sp))- UfsusCfsusAfsgsAfscsCfsusGfsusUfsusUfsgsCfsusUfsdTsdT PCSK9(21) 121 (All (Rp))- UfsuCfsuAfsgAfscCfsuGfsuUfsuUfsgCfsuUfsdTsdT PCSK9(22) 121 (All (Sp))- UfsuCfsuAfsgAfscCfsuGfsuUfsuUfsgCfsuUfsdTsdT PCSK9(23) 121 (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp, Rp, Sp, Rp, Sp)-UfsusCfsusAfsgsAfscsCfsusGfsusUfsusUfsgsCfsusUfsdTsdT PCSK9 (24) 121(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp, Rp)- UfsusCfsusAfsgsAfscsCfsusGfsusUfsusUfsgsCfsusUfsdTsdT NOTE:lower case letters represent 2′-OMe RNA residues; capital lettersrepresent 2′-F RNA residues; d = 2′-deoxy residues; and “s” indicates aphosphorothioate moiety.

Synthesis examples for Human PCSK9 siRNA fully modified 2′-F/2′-OMeAntisense Strands having several chiral phosphorothioate internucleotidelinkages and full chiral phosphorothioate internucleotide linkages.

SEQ ID NO: Human PCSK9 siRNA fully modified 2′-F/2′-OMe AntisenseStrands PCSK9 (25) 122 (All (Rp))-asAfsgsCfsasAfsasAfscsAfsgsGfsusCfsusAfsgsAfsasdTsdT PCSK9 (26) 122 (All(Sp))- asAfsgsCfsasAfsasAfscsAfsgsGfsusCfsusAfsgsAfsasdTsdT PCSK9 (27)122 (All (Rp))- asAfgCfaAfaAfcsAfsgsGfsusCfsusAfsgsAfsasdTsdT PCSK9 (28)122 (All (Sp))- asAfgCfaAfaAfcsAfsgsGfsusCfsusAfsgsAfsasdTsdT PCSK9 (29)122 (All (Rp))- asAfsgCfsaAfsaAfscAfsgGfsuCfsuAfsgAfsadTsdT PCSK9 (30)122 (All (Sp))- asAfsgCfsaAfsaAfscAfsgGfsuCfsuAfsgAfsadTsdT PCSK9 (31)122 (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp)-asAfgCfaAfasAfscAfsgsGfsusCfsusAfsgsAfsasdTsdT PCSK9 (32) 122 (Sp, Rp,Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp)-asAfgCfaAfasAfscAfsgsGfsusCfsusAfsgsAfsasdTsdT NOTE: lower case lettersrepresent 2′-OMe RNA residues; capital letters represent 2′-F RNAresidues; d = 2′-deoxy residues; and “s” indicates a phosphorothioatemoiety.

To assemble duplexes, RNA strand thermal annealing and preparation ofsiRNA duplexes is performed. Specifically, each RNA strand is mixed withits complementary RNA strand in equimolar concentration of 10 μM in1×PBS. Total 0.5 mL solution is prepared for each duplex and the mixtureis heated at 90° C. for 2 min and is allowed to cool down over thecourse of several hours. The mixtures are then stored at 4° C.

Following the thermal RNA strand annealing step, all the possible siRNAduplex combinations can be prepared by annealing any of the Sensestrands with any possible complementary strand of the Antisense strands.

All prepared siRNA duplexes can be evaluated in vitro for their PCSK9gene-silencing properties, following transfection in HeLa cells or Hep3Bcells (e.g., as described herein). According to the present invention,different potencies may be observed for different duplexes, e.g., thatvary in number, position, and/or stereo architecture of the chiralphosphorothioate backbone linkages, and optionally also in presence,level, and or type of one or more other chemical modifications.

Any or all siRNA properties such as: nuclease resistance, cellpenetration, endosomal escape, duplex thermodynamic stability,tridemnsional structure of the duplex, affinity towards the variousmechanistic steps of enzyme interactions, affinity towards the targetmRNA, specific off-target effects, immunostimulation, duration ofaction, pharmacokinetics, etc. may be modulated and influenced by thestereochemistry of the chiral phosphorothioate backbone linkages, asdescribed herein.

Example 64. Preparation of Chirally Controlled Preparations ofOligonucleotide

The present Example describes preparation of a variety of particularchirally controlled compositions of certain oligonucleotides describedtherein.

N⁴-Acetyl-5′-O-DMTr-2′-O-methylcytidine (1) (1.15 g, 1.91 mmol) wasdissolved in anhydrous DCM (20 mL) and mixed with 2 equiv of succinicanhydride (0.383 g, 3.82 mmol) and 3 equiv of4-N,N-dimethylaminopyridine (0.701 g, 5.73 mmol). The reaction wasstirred under Argon at room temperature. After complete consumption ofstarting material as determined by TLC (1 hour), the solvents wereevaporated to dryness, the crude residue was dissolved in DCM containing1% triethylamine then purified by flash silica gel chromatography usinga gradient of 0-2% of MeOH in DCM containing 2% of triethylamine. Yieldof pure succinyl compound (2) after evaporation was 1.50 g, 98%. MS (ESI+ve): calc (M+H)⁺: 702.27, found: 702.34. The resultingN⁴-acetyl-5′-O-DMTr-3′-O-succinyl-2′-O-methylcytidine (2) (0.18 g, 0.22mmol), N,N-diisopropylethylamine (0.18 mL, 0.79 mmol) and GE CustomSupport™ Amino (1 g) were taken up in DMF (5 mL) then HBTU (0.10 g, 0.26mmol) was added. The mixture was shaken for 2 h then filtered. Thesupport was washed with DMF, MeOH and finally DCM then dried in vacuo.Trityl cation analysis (monitoring at 504 nm) showed that the loading ofnucleoside on the support (3) was 180 μmol/g.

Example 65. Preparation of Chirally Controlled Preparations ofOligonucleotide

Using a procedure analogous to that described in Example 64,N²-Phenoxyacetyl-5′-O-DMTr-3′-O-succinyl-2′-O-methylguanosine (4, MS(ESI +ve): calc (M+H)⁺: 834.30, found: 834.32) was loaded onto GE CustomSupport™ Amino. Trityl cation analysis (monitoring at 504 nm) showedthat the loading of nucleoside on the support (6) was 140 μmol/g.

In some embodiments, oligonucleotides containing stereodefined 2′-OMephosphorothioate triester internucleotidic linkages was synthesized onABI 394 DNA/RNA synthesizer according to the cycle summarized in TableE-27 using a 10 μmol capacity synthesis column loaded with 60 mg (10.8and 8.4 μmol respectively) of either uncapped succinyl linked5′-O-DMTr-2′-O-methyl-G_(Pac) (6, 140 μmol/g) or5′-O-DMTr-2′-O-methyl-C^(Ac) (3, 180 μmol/g). The synthesis cycle wasperformed with a preliminary capping step (capping 2) and with removalof the terminal 5′-O-DMTr group. The stereospecific sulfurization stepswere performed using the 0.3 M S-(2-cyanoethyl)methylthiosulfonatereagent in ACN containing BSTFA, following the coupling of thecorresponding chiral phosphoramide and the two-step capping process(Table E-27).

Once the automated oligonucleotide synthesis cycle was complete, and thefinal 5′-O-DMTr group removed, the synthesis column was taken off theDNA/RNA synthesizer and dried under vacuum. The dried support wastransferred onto an empty glass manual peptide synthesizer and 10 mLsolution of 0.5 M 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 0.25 MN,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in ACN wascontinuously added to the support for 5 min without stopping the flow inthe manual peptide synthesizer. The support was washed by ACN and driedin vacuo. Then, the support was treated with 5% EtOH/conc NH₃ (5 mL) at60° C. for 6 h, and left at room temperature for 12 h. The support wasremoved by filtration and washed with conc. NH₃. The filtrate wasconcentrated in vacuo then purified by IEX.

TABLE E-27 Summary for Oligonucleotide Synthesis. step reaction reagentdelivery time (sec) wait time (sec) 1 detritylation 3% TCA in DCM 3 +120 + 10 N.A. 2 coupling 0.15M 8 + 6 30 + 900 phosphoramidite in ACN +2M CMPT in ACN 3 capping 1 5% Pac₂O in THF/2,6- 30 60 lutidine 4 capping2 5% Pac₂O in THF/2,6- 30 60 lutidine + 16% NMI in THF 5 sulfurization0.3M (S-cyanoethyl)- 15 + 3 × 4 120 + 3 × 60 + 300 methylthiosulfonate

Synthesis of Oligonucleotide ONT-94: (All (Sp))-gsgsusgsgsasasgsgsc (SEQID NO: 124). t_(R) (IEX-HPLC): 18.26 min. Calc MW: 3563.9; Found MW:3562.6.

Synthesis of Oligonucleotide ONT-96: (All (Rp))-gsgsusgsgsasasgsgsc (SEQID NO: 124). t_(R) (IEX-HPLC): 18.16 min. Calc MW: 3563.9; Found MW:3561.7.

Synthesis of Oligonucleotide ONT-98: (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp) -gsgsusgsgsasasgsgsc (SEQ ID NO: 124). t_(R) (IEX-HPLC): 18.05 min.Calc MW: 3563.9; Found MW: 3562.5.

Synthesis of Oligonucleotide ONT-100: (Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp) -gsgsusgsgsasasgsgsc (SEQ ID NO: 124). t_(R) (IEX-HPLC): 17.86 min.Calc MW: 3563.9; Found MW: 3561.1.

Synthesis of Oligonucleotide ONT-102: (Rp, Rp, Sp, Sp, Sp, Sp, Sp, Rp,Rp) -gsgsusgsgsasasgsgsc (SEQ ID NO: 124). t_(R) (IEX-HPLC): 18.30 min.Calc MW: 3563.9; Found MW: 3561.3.

Synthesis of Oligonucleotide ONT-104: (Sp, Sp, Rp, Rp, Rp, Rp, Rp, Sp,Sp)-gsgsusgsgsasasgsgsc (SEQ ID NO: 124). t_(R) (IEX-HPLC): 17.95 min.Calc MW3563.9; Found MW: 3562.7.

Synthesis of Oligonucleotide ONT-95: (All (Sp))-gscscsuscscsasg. t_(R)(IEX-HPLC): 14.78 min. Calc MW: 2709.2; Found MW: 2707.4.

Synthesis of Oligonucleotide ONT-97: (All (Rp))-gscscsuscscsasg. t_(R)(IEX-HPLC): 15.60 min. Calc MW: 2709.2; Found MW: 2708.3.

Synthesis of Oligonucleotide ONT-99: (Rp, Sp, Rp, Sp, Rp, Sp,Rp)-gscscsuscscsasg. t_(R) (IEX-HPLC): 16.10 min. Calc MW: 2709.2; FoundMW: 2708.0.

Synthesis of Oligonucleotide ONT-101: (Sp, Rp, Sp, Rp, Sp, Rp,Sp)-gscscsuscscsasg. t_(R) (IEX-HPLC): 16.23 min. Calc MW: 2709.2; FoundMW: 2708.2.

Synthesis of Oligonucleotide ONT-103: (Rp, Rp, Sp, Sp, Sp, Rp,Rp)-gscscsuscscsasg. t_(R) (IEX-HPLC): 16.26 min. Calc MW: 2709.2; FoundMW: 2707.8.

Synthesis of Oligonucleotide ONT-105: (Sp, Sp, Rp, Rp, Rp, Sp,Sp)-gscscsuscscsasg. t_(R) (IEX-HPLC): 16.22 min. Calc MW: 2709.2; FoundMW: 2710.0.

Oligonucleotides containing stereodefined chimeric 2′-OMephosphorothioate triester and 2′-O-MOE phosphorothioate triesterinternucleotidic linkages was synthesized on ABI 394 DNA/RNA in ananalogous fashion to those described in examples herein.

Synthesis of Oligonucleotide ONT-90: (All(Rp))-G_(MOE)sG_(MOE)susG_(MOE)sG_(MOE)sasasG_(MOE)sG_(MOE)sc (SEQ IDNO: 124). t_(R) (IEX-HPLC): 15.35 min. Calc MW: 3828.2; Found MW:3826.5.

Synthesis of Oligonucleotide ONT-119: (All(Sp))-G_(MOE)sG_(MOE)susG_(MOE)sG_(MOE)sasasG_(MOE)sG_(MOE)sc (SEQ IDNO: 124). t_(R) (IEX-HPLC): 16.42 min. Calc MW: 3828.2; Found MW:3827.2.

Synthesis of Oligonucleotide ONT-91: (All (Rp))-G_(MOE)scscsuscscsasg.t_(R) (IEX-HPLC): 15.69 min. Calc MW: 2753.3; Found MW: 2751.5.

Synthesis of Oligonucleotide ONT-120: (All (Sp))-G_(MOE)scscsuscscsasg.t_(R) (IEX-HPLC): 14.71 min. Calc MW: 2753.3; Found MW: 2751.4.

Example 66. General IEX-HPLC Method for the Analysis of Crude andPurified DMT Off RIPtide Oligonucleotides Buffer A: 10 mM TrisHCl, 50%ACN, pH 8.0 Buffer B: 10 mM TrisHCl, 800 mM NaClO₄, 50% ACN, pH 8.0Column: DIONEX, DNAPac, PA-200, Analytical, 4.0×250 mm

Column temperature=60° C.Signal monitored at 254 and 280 nmGradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 95 5 2 1 95 5 1 22 1 7030 6 25 1 5 95 6 25.5 1 95 5 6 30 1 95 5 1

Example 67. General UPLC-LCMS Method for the Analysis of Purified DMTOff RIPtide Oligonucleotides Buffer A: 15 mM TEA, 400 mM HFIP, WaterBuffer B: 50:50 Buffer A/Methanol Column: UPLC@OST C₁₈ 1.7 μm, 2.1×500mm

Column temperature=50° C.Gradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 0.2 80 20 2 0.2 80 20 122 0.2 30 70 6 25 0.2 5 95 6 25.5 0.2 80 20 6 30 0.2 80 20 1

Example 68. General IEX-HPLC Method for the Purification of Crude DMTOff RIPtide Oligonucleotide Buffer A: 20 mM NaOH, pH 11.0 Buffer B: 20mM NaOH, 2.5 M NaCl, pH 11.0

Column: Empty column Waters AP-1 (Waters), custom in-house packed withSource 15Q support (GE Healthcare). The same purification column wasused for the different stereopure RIPtide oligonucleotides.Instrument: AKTA Purifier, equipped with the P-900 pump, the UPC-900detector and a 50 mL injection SuperLoop (GE Healthcare)Buffer heater temperature set=70° C.Column heater tape set=70° C.Signal monitored at 254 nmFractions volume: 5 mLGradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 100 0 15 4 100 0 1 25 490 10 6 35 4 90 10 1 45 4 80 20 6 60 4 80 20 1 80 4 65 35 6 95 4 65 35 1120 4 45 55 6 125 4 45 55 1 126 4 100 0 6 140 4 100 0 1

Collected fractions were individually analyzed by analytical IEX-HPLCusing the analytical conditions and gradient described above. Purefractions were pooled in order to provide purified material of 95% andabove purity as determined by the 254 nm UV absorbance profiles.

General RP-Sep-Pak method for the desalting of purified RIPtidesoligonucleotides. The solution of pooled pure fractions was loaded on aSep-Pak cartridge (Waters, Sep-Pak Vac 35 cc (10 g) C₁₈ Cartridges)pre-conditioned with water. After loading of the sample (100 mL), thecartridge was washed with milli Q water (50 mL) to remove all salt andthen washed with 50% ACN/water to elute the full length desalted RNAoligonucleotide. The collected solution was concentrated in vacuo to avolume of 5 mL and lyophilized from water.

A panel of stereocontrolled fully phosphorothioate modified RIPtides(8-mer or 10-mer) that bind naked hTR are investigated for the in vitroinhibition of the activity of the telomerase RNP complex. The Cy5-TRAPassay (Shay et al., Nat. Protoc., 2006, 1, 1583), a variation of theTRAP (Shay et al., Science, 1994, 266, 2011), is used to the determinethe IC₅₀ values for the stereocontrolled phosphorothioate RIPtides usingHeLa cell extracts, according to previously reported protocols (Verdineet al., J. Biol. Chem., 2012, 287, 18843).

TRAP assays are performed following previously reported protocols thatuse fluorescence as a quantitation system, with some modifications.Briefly, extension of a fluorescent artificial substrate by telomeraseis carried out for 30 minutes at 30° C., followed by amplification with30 PCR cycles (34° C. 30 s, 59° C. 30 s, 72° C. 1 min). The inhibitorypotential of the RIPtides is initially assessed in HeLa cell extracts,in duplicate experiments, using a 600 μM-60 μM concentration range.Experiments with selected RIPtides are repeated for a concentrationrange of 0.06 μM-60 μM in HeLa, DU145 (prostate cancer) and HEK293 cellextracts. Several controls are used in these assays: a positive control(untreated cell lysate), negative controls (buffer only, heatinactivated and RNase treated cell extracts), and PCR amplificationcontrol (60 μM of RIPtide added after telomerase elongation and beforePCR). In certain embodiments, chirally controlled RIPtide preparationsshow enhanced inhibition of hTR as compared with the positive controland/or enhanced inhibition of hTRas compared with the negative control;in some embodiments, some, but optionally not all RIPtides of a givensequence show such properties. In some embodiments, by “enhanced” inthis context is meant enhanced by between about five-fold and aboutten-fold. In some embodiments, by “enhanced” is meant at least about1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, 9.5, or 10.0-fold.

Cell Culturing Conditions. The transformed embryonic kidney cell lineHEK293 and the prostate cancer cell line DU145 are maintained in DMEMsupplemented with 10% fetal bovine serum in 5% CO₂ at 37° C. Solublecell extracts for TRAP assays are prepared by detergent lysis of 106cells with 200 μL 1×CHAPS Lysis Buffer (Chemicon) as described in themanufacturer's instructions.

According to the present invention, different potencies and/or differentother properties may be observed for the various stereoarchitectures offully stereocontrolled phosphorothioate RIPtides. For instance, RIPtideproperties such as: nuclease resistance, tissue accumulation, cellpenetration, endosomal escape, tridemnsional structure of the RIPtide,affinity towards the target folded hTR RNA, immunostimulation, durationof action, pharmacokinetics, etc. may vary for chirally controlledRIPtide preparations that share the same sequence but differ from oneanother with respect to different location and/or stereochemistry of oneor more chiral phosphorothioate backbone linkages.

Example 69: Chirally Controlled Oligonucleotide Compositions ShowDifferent Activity In Vivo as Compared with Chirally UncontrolledCompositions Having the Same Sequence

The present Example compares in vivo pharmacological activity ofchirally pure oligonucleotides with that observed for the “parent”stereorandom mixture (i.e., for a composition containingoligonucleotides of the same sequence as the chirally pureoligonucleotides but not displaying chiral purity, for example as aresult of having been prepared via a stereorandom process). Fourchirally pure oligonucleotides, each of which had a sequencecomplementary to that of a particular target transcript or gene encodinga protein of interest were synthesized, formulated, and administeredtwice per week for 5 weeks at two dose levels each, to animalsexpressing the target gene. Levels of encoded protein levels werequantified. In this Example, oligonucleotides having a sequenceantisense to (and therefore targeting) human Apolipoprotein-B (ApoB)were used for proof-of-concept in transgenic mice expressing human ApoB.

Test Articles

Test articles were PBS alone (i.e., no oligonucleotide control) or therelevant oligonucleotide composition (i.e., Mipomersen (ONT-41), ONT-75,ONT-77, ONT-80, or ONT-81 oligonucleotides (see Example 52 forstructures) were formulated in 1×PBS (diluted from 10×PBS (LifeTechnologies, AM9624) using nuclease-free water (Qiagen, 129115)) at 0.5and 1 mg/mL for dosing at 5 and 10 mg/kg, respectively. Formulationswere based on absolute mass with no adjustment for the active material.Oligonucleotide concentrations were confirmed by measurement with aCarry-100 UV-Vis (Agilent Technologies). Samples of all test articleswere checked for endotoxin levels using a kinetic pyrochrome chromogenicendotoxin assay (Associates of Cape Cod, 1500-5, E005-5). All testedsamples were found to have a lower amount of endotoxin than the 0.5EU/ml acceptable limit.

Animals and In Vivo Procedures

Female transgenic mice (huApoB mice) expressing high plasmaconcentrations of human apolipoprotein B100 and lipoprotein(a) (J. Clin.Invest. 92: 3029-3037) were obtained from Taconic (strainB6.SJL-Tg(APOB)|102Sgy N20+?, model #1004-F). All animals were genotypedprior to delivery.

Mice were delivered and allowed to acclimate for at least seven daysprior to study start. All mice were given regular chow and water adlibitum, and were not fasted prior to compound administration. Mice wererandomized to study groups, and dosed intraperitoneally (IP) at 10 ml/kgon Days 1, 4, 8, 11, 15, 18, 22, 25, 29 and 32, based on individualmouse body weight measured prior to dosing on each dosing day. Blood wascollected during the course of the study on Days 0 (day before firstdose), 17, 24, 31, 38, 45, 52 and 60 by submandibular (cheek) bleed andat sacrifice on Day 66 by cardiac puncture, and then processed to serum.

Apolipoprotein B Protein Assay

ApoB protein levels in serum were measured using an ApoB Human ELISA Kit(Abcam, ab108807). Serum samples were diluted 1:20,000 and assayed perthe kit recommended protocol without modifications. Automated washeswere done with 30 second soak periods using an Aquamax 4000 (MolecularDevices). Reactions were stopped after 12 minute incubations withChromogen substrate and measurements were taken with a Spectramax M5(Molecular Devices).

Standard curves were generated by plotting the standard concentration onthe x-axis and the corresponding mean 450 nm absorbance on the y-axis.The best-fit line was determined by regression analysis using log-logcurve-fit. Each assay plate included a negative (PBS treated) and apositive (Mipomersen-treated) control and each mouse serum sample wasassayed in 4 technical replicates.

For each sample, the mean absolute level of ApoB was normalized to themean value for the PBS treated group, to obtain the relative level ofApoB protein expression. In one instance, an animal was excluded fromthe analysis as measurements deviated from the cohort mean by 2 standarddeviations. Relative levels of ApoB protein expression were used forstatistical analysis (Graphpad Prism) by 2-way ANOVA followed byNewman-Keuls post-hoc test.

Results

The results are shown in Table E-28a and FIG. 40. Relative to PBS, 5mg/kg IP ONT-75 resulted in significant reductions of serum ApoB proteinlevels on day 24 of the study, to 87% (p<0.05). Relative to PBS, 5 mg/kgIP ONT-77 resulted in significant reductions of serum ApoB proteinlevels on days 24, and 31 of the study, to 72% (p<0.0001), 81% (p<0.05)of control levels, respectively. Relative to PBS, 5 mg/kg IP ONT-80resulted in significant reductions of serum ApoB protein levels on days24 and 31 of the study, to 83% (p<0.05), 80% (p<0.05) of control levels,respectively. Relative to PBS, 5 mg/kg IP ONT-81 resulted in significantreductions of serum ApoB protein levels on days 17 and 24 of the study,to 85% (p<0.05), 70% (p<0.0001) of control levels, respectively.

TABLE E-28a Serum Human Apolipoprotein B Levels Relative to PBS AfterMultiple IP Doses of 5 mg/kg Stereioisomer or Mipomersen in huApoB Mice(N = 4-5] Day Sample 17 24 31 38 45 PBS 100 ± 9  100 ± 9 100 ± 9   100 ±11  100 ± 9  ONT-41   54 ± 11  49 ± 7 54 ± 9   64 ± 10  80 ± 9 ONT-75103^(d) ± 11 87^(dw) ± 10 94^(d) ± 10 97^(d) ± 10 98^(a) ± 9 ONT-77 83^(d) ± 15  72^(cz) ± 10 81^(dw) ± 9  91^(d) ± 13  95^(a) ± 12 ONT-8089^(d) ± 9 83^(dw) ± 8  80^(dw) ± 3  95^(d) ± 7   87 ± 11 ONT-81 85^(dw)± 12 70^(cz) ± 5  88^(d) ± 15 101^(d) ± 10  102^(b) ± 7 ^(a)Statistically different from the Mipomersen control group (ONT-41),with p < 0.05 (2-way ANOVA with Newman-Keuls post-hoc test)^(b)Statistically different from the Mipomersen control group (ONT-41),with p < 0.01 (2-way ANOVA with Newman-Keuls post-hoc test)^(c)Statistically different from the Mipomersen control group (ONT-41),with p < 0.001 (2-way ANOVA with Newman-Keuls post-hoc test)^(d)Statistically different from the Mipomersen control group (ONT-41),with p < 0.0001 (2-way ANOVA with Newman-Keuls post-hoc test)^(w)Statistically different from the PBS control group, with p < 0.5(2-way ANOVA with Newman-Keuls post-hoc test) x: Statistically differentfrom the PBS control group, with p < 0.01 (2-way ANOVA with Newman-Keulspost-hoc test) y: Statistically different from the PBS control group,with p < 0.001 (2-way ANOVA with Newman-Keuls post-hoc test)^(z)Statistically different from the PBS control group, with p < 0.0001(2-way ANOVA with Newman-Keuls post-hoc test)

Compared with 5 mg/kg IP Mipomersen, ONT-75 administered with the samedosing paradigm resulted in significantly less reduction of serum ApoBprotein levels on days 17, 24, 31 and 38 (p<0.0001), and 45 (p<0.05) ofthe study. Compared with 5 mg/kg IP Mipomersen, ONT-77 administered withthe same dosing paradigm resulted in significantly less reduction ofserum ApoB protein levels on days 17, 31 and 38 (p<0.0001), 24 (p<0.001)and 45 (p<0.05) of the study. Compared with 5 mg/kg IP Mipomersen,ONT-80 administered with the same dosing paradigm resulted insignificantly less reduction of serum ApoB protein levels on days 17,24, 31 and 38 (p<0.0001), but was not different on day 45 (p>0.05) ofthe study. Compared with 5 mg/kg IP Mipomersen, ONT-81 administered withthe same dosing paradigm resulted in significantly less reduction ofserum ApoB protein levels on days 17, 31 and 38 (p<0.0001), 24 (p<0.001)and 45 (p<0.01) of the study. FIG. 40 shows the timecourse of SerumHuman Apolipoprotein B protein levels relative to PBS after 5 mg/kgstereoisomer or mipomersen IP dosing in huApoB mice. Downward arrowsindicate dosing days. Group means normalized to the PBS control groupare shown, where each groups comprised 4-5 animals. Error bars representstandard deviations.

The results of the 10 mg/kg dosing paradigm are shown in Table E-28b andFIG. 53. Relative to PBS, 10 mg/kg IP ONT-75 resulted in significantreductions of serum ApoB protein levels on days 17, 24 and 38 of thestudy, to 78% (p<0.0001), 76% (p<0.0001) and 82% (p<0.001),respectively. Relative to PBS, 10 mg/kg IP ONT-77 resulted insignificant reductions of serum ApoB protein levels on days 17, 24, 31,38, 45 and 52 of the study, to 62% (p<0.0001), 61% (p<0.0001), 54%(p<0.0001), 59% (p<0.0001), 72% (p<0.0001), and 73% (p<0.0001) ofcontrol levels, respectively. Relative to PBS, 10 mg/kg IP ONT-80resulted in significant reductions of serum ApoB protein levels on days17, 24, 31, 38, 45, 52 and 66 of the study, to 55% (p<0.0001), 59%(p<0.0001), 73% (p<0.0001), 67% (p<0.0001), 76% (p<0.0001), 76%(p<0.0001), and 80% (p<0.01) of control levels, respectively. Relativeto PBS, 10 mg/kg IP ONT-81 resulted in significant reductions of serumApoB protein levels on days 17, 24, 31, 38 and 45 of the study, to 49%(p<0.0001), 62% (p<0.0001), 71% (p<0.0001), 74% (p<0.0001) and 79%(p<0.001) of control levels, respectively.

TABLE E-28b Serum Human Apolipoprotein B Levels Relative to PBS AfterMultiple IP Doses of 10 mg/kg Stereioisomer (ONT-75, -77, -80 or -81) orMipomersen in huApoB Mice (N = 4-5) Day Sample 17 24 31 38 45 52 60 66PBS  100 ± 11  100 ± 9  100 ± 17  100 ± 11 100 ± 7  100 ± 7  100 ± 9 100 ± 7  ONT-41   31 ± 13   24 ± 24  23 ± 6  29 ± 10  46 ± 10  64 ± 8 77± 8  64 ± 7 ONT-75 78^(dz) ± 5 76^(dz) ± 4 82^(dy) ± 7 95^(d) ± 9 97^(d) ± 3   94^(d) ± 13 108^(d) ± 6    87^(c) ± 12 ONT-77 62^(dz) ± 961^(dz) ± 4 54^(dz) ± 6 59^(dz) ± 10 72^(dz) ± 7  73^(z) ± 7 95^(a) ± 5  87^(b) ± 11 ONT-80 55^(cz) ± 4 59^(dz) ± 3 73^(dz) ± 4 67^(dz) ± 1076^(dz) ± 10  76^(z) ± 11  87 ± 10 80^(bx) ± 7  ONT-81 49^(bz) ± 7 62^(dz) ± 19 71^(dz) ± 7 74^(dz) ± 7  79^(dy) ± 11 88^(c) ± 5 90 ± 689^(c) ± 8 ^(a)Statistically different from the Mipomersen control group(ONT-41), with p < 0.05 (2-way ANOVA with Newman-Keuls post-hoc test)^(b)Statistically different from the Mipomersen control group (ONT-41),with p < 0.01 (2-way ANOVA with Newman-Keuls post-hoc test)^(c)Statistically different from the Mipomersen control group (ONT-41),with p < 0.001 (2-way ANOVA with Newman-Keuls post-hoc test)^(d)Statistically different from the Mipomersen control group (ONT-41),with p < 0.0001 (2-way ANOVA with Newman-Keuls post-hoc test) w:Statistically different from the PBS control group, with p < 0.05 (2-wayANOVA with Newman-Keuls post-hoc test) ^(x)Statistically different fromthe PBS control group, with p < 0.01 (2-way ANOVA with Newman-Keulspost-hoc test) ^(y)Statistically different from the PBS control group,with p < 0.001 (2-way ANOVA with Newman-Keuls post-hoc test)^(z)Statistically different from the PBS control group, with p < 0.0001(2-way ANOVA with Newman-Keuls post-hoc test)

Compared with 10 mg/kg IP Mipomersen, ONT-75 administered with the samedosing paradigm resulted in significantly lower reduction (knockdown) ofserum ApoB protein levels on days 17, 24, 31, 38, 45, 52, 60 (p<0.0001),and 66 (p<0.001) of the study. Compared with 10 mg/kg IP Mipomersen,ONT-77 administered with the same dosing paradigm resulted insignificantly lower reduction (knockdown) of serum ApoB protein levelson days 17, 24, 31, 38, and 45 (p<0.0001), 60 (p<0.05) and 66 (p<0.01)of the study. Compared with 10 mg/kg IP Mipomersen, ONT-80 administeredwith the same dosing paradigm resulted in significantly lower reduction(knockdown) of serum ApoB protein levels on days 24, 31, 38 and 45(p<0.0001), 17 (p<0.001) and 66 (p<0.01). Compared with 10 mg/kg IPMipomersen, ONT-81 administered with the same dosing paradigm resultedin significantly lower reduction (knockdown) of serum ApoB proteinlevels on days 24, 31, 38 and 45 (p<0.0001), 17 (p<0.01), 52 and 66(p<0.001) of the study.

By day 38 (i.e. 6 days after the last dose), serum ApoB protein levelshad returned to baseline after ONT-75. Considering the initial serumApoB protein level reduction, the rate of return to baseline of serumApoB protein was slower after ONT-77 and ONT-80 and the level of serumApoB protein is similar to Mipomersen levels by day 52.

The results of the 5 mg/kg dosing paradigm of additional stereopurearchitectures are shown in Table E-28c and FIG. 54. Relative to PBS, 5mg/kg IP ONT-87 resulted in significant reductions of serum ApoB proteinlevels on days 17, 24, 31 and 38 of the study, to 27% (p<0.0001), 40%(p<0.0001), 55% (p<0.0001) and 71% (p<0.0001) of control levels,respectively. Relative to PBS, 5 mg/kg IP ONT-88 resulted in significantreductions of serum ApoB protein levels on days 17, 24, 31 and 38 of thestudy, to 47% (p<0.0001), 34% (p<0.0001), 69% (p<0.0001) and 85%(p<0.0001) of control levels, respectively. Relative to PBS, 5 mg/kg IPONT-89 resulted in significant reductions of serum ApoB protein levelson days 17, 24 and 31 of the study, to 43% (p<0.0001), 48% (p<0.0001)and 85% (p<0.05) of control levels, respectively.

TABLE E-28c Serum Human Apolipoprotein B Levels Relative to PBS AfterMultiple IP Doses of 5 mg/kg Stereioisomer (ONT-87, -88 or -89) orMipomersen in huApoB Mice (N = 3-4) Day SAMPLES 17 24 31 38 PBS 100 ± 5  100 ± 12 100 ± 4  100 ± 8  ONT-41 33^(z) ± 3 44^(z) ± 2  75^(y) ± 1176^(y) ± 8 ONT-87  27^(z) ± 11 40^(z) ± 5 55^(bz) ± 8   71^(z) ± 7ONT-88 47^(az) ± 5  34^(z) ± 8  69^(z) ± 13 85^(z) ± 7 ONT-89 43^(z) ± 948^(z) ± 6 85^(w) ± 12 93^(a) ± 7 ^(a)Statistically different from theMipomersen control group (ONT-41), with p < 0.05 (2-way ANOVA withNewman-Keuls post-hoc test) ^(b)Statistically different from theMipomersen control group (ONT-41), with p < 0.01 (2-way ANOVA withNewman-Keuls post-hoc test) c: Statistically different from theMipomersen control group (ONT-41), with p < 0.001 (2-way ANOVA withNewman-Keuls post-hoc test) d: Statistically different from theMipomersen control group (ONT-41), with p < 0.0001 (2-way ANOVA withNewman-Keuls post-hoc test) ^(w)Statistically different from the PBScontrol group, with p < 0.05 (2-way ANOVA with Newman-Keuls post-hoctest) x: Statistically different from the PBS control group, with p <0.01 (2-way ANOVA with Newman-Keuls post-hoc test) ^(y)Statisticallydifferent from the PBS control group, with p < 0.001 (2-way ANOVA withNewman-Keuls post-hoc test) ^(z)Statistically different from the PBScontrol group, with p < 0.0001 (2-way ANOVA with Newman-Keuls post-hoctest)

Compared with 5 mg/kg IP Mipomersen, ONT-87 administered with the samedosing paradigm resulted in significantly higher reduction (knock-down)of serum ApoB protein levels on day 31, (p<0.01) of the study. Comparedwith 5 mg/kg IP Mipomersen, ONT-88 administered with the same dosingparadigm resulted in significantly lower reduction (knock-down) of serumApoB protein levels on day 17 (p<0.05 of the study). Compared with 5mg/kg IP Mipomersen, ONT-89 administered with the same dosing paradigmresulted in significantly lower reduction (knockdown) of serum ApoBprotein levels on day 38 (p<0.05) of the study.

The results of the 10 mg/kg dosing paradigm of additional stereopurearchitectures are shown in Table E-28d and FIGS. 55 and 56. Relative toPBS, 10 mg/kg IP ONT-87 resulted in significant reductions of serum ApoBprotein levels on days 17, and 24 of the study, to 27% (p<0.0001), 14%(p<0.0001) of control levels, respectively. Relative to PBS, 10 mg/kg IPONT-88 resulted in significant reductions of serum ApoB protein levelson days 17, and 24 of the study, to 23% (p<0.0001), and 17% (p<0.0001)of control levels, respectively. Relative to PBS, 10 mg/kg IP ONT-89resulted in significant reductions of serum ApoB protein levels on days17, and 24 of the study, to 29% (p<0.0001), and 23% (p<0.0001) ofcontrol levels, respectively.

TABLE E-28d Serum Human Apolipoprotein B Levels Relative to PBS AfterMultiple IP Doses of 10 mg/kg Stereioisomer (ONT-87, -88 or -89) orMipomersen in huApoB Mice (N = 3-4) Day Sample 17 24 PBS 100 ± 5  100 ±8  ONT-41  44^(z) ± 17 27^(z) ± 9 ONT-87 27^(az) ± 10 14^(z) ± 3 ONT-8823^(az) ± 6  17^(z) ± 5 ONT-89 29^(az) ± 12 23^(z) ± 8 ^(a)Statisticallydifferent from the Mipomersen control group (ONT-41), with p < 0.05(2-way ANOVA with Newman-Keuls post-hoc test) b: Statistically differentfrom the Mipomersen control group (ONT-41), with p < 0.01 (2-way ANOVAwith Newman-Keuls post-hoc test) c: Statistically different from theMipomersen control group (ONT-41), with p < 0.001 (2-way ANOVA withNewman-Keuls post-hoc test) d: Statistically different from theMipomersen control group (ONT-41), with p < 0.0001 (2-way ANOVA withNewman-Keuls post-hoc test) w: Statistically different from the PBScontrol group, with p < 0.05 (2-way ANOVA with Newman-Keuls post-hoctest) x: Statistically different from the PBS control group, with p <0.01 (2-way ANOVA with Newman-Keuls post-hoc test) y: Statisticallydifferent from the PBS control group, with p < 0.001 (2-way ANOVA withNewman-Keuls post-hoc test) ^(z)Statistically different from the PBScontrol group, with p < 0.0001 (2-way ANOVA with Newman-Keuls post-hoctest)

Compared with 10 mg/kg IP Mipomersen, ONT-87 administered with the samedosing paradigm resulted in significantly higher reduction (knockdown)of serum ApoB protein levels on day 17, (p<0.05) of the study. Comparedwith 10 mg/kg IP Mipomersen, ONT-88 administered with the same dosingparadigm resulted in significantly higher reduction (knockdown) of serumApoB protein levels on day 17 (p<0.05) of the study. Compared with 10mg/kg IP Mipomersen, ONT-89 administered with the same dosing paradigmresulted in significantly higher reduction (knockdown) of serum ApoBprotein levels on day 17 (p<0.05) of the study.

Thus, in at least some embodiments, the present invention provideschirally controlled oligonucleotide compositions that show a biologicalactivity and are characterized by extended persistence and/or slowerrate of decay of that activity over time as compared with an appropriatereference (e.g., a preparation of oligonucleotides of the same sequencebut different chiral specificity, including particularly stereorandompreparations). In some embodiments where such extended persistenceand/or slower rate of decay is observed, provided chirally controlledcompositions may be administered according to a dosing regimen withfewer total doses and/or longer periods between two or more doses thatis utilized with the “parent” stereorandom composition to achievecomparable biologic and/or therapeutic effect. For example, the slowerreturn to baseline of serum ApoB protein following treatment withchirally pure oligonucleotide compared with Mipomersen as demonstratedin the present Example, suggests that chirally pure ApoBoligonucleotides can be dosed less frequently than the parentalstereorandom mixture (e.g., than Mipomersen).

The results presented in this Example demonstrate, for instance, thatchirally pure oligonucleotide compositions can have significantlydifferent pharmacological activity in vivo as compared with anappropriate reference (e.g., a preparation of oligonucleotides of thesame sequence but different chiral specificity, including particularlystereorandom preparations), and specifically as compared with a“parental” stereorandom preparation. Those skilled in the art, in lightof this demonstration, will appreciate that chirally controlledoligonucleotide compositions provided by the present disclosure haveunexpected activities and characteristics. Those skilled in the art, inlight of the present disclosure, will further appreciate that variousmethodologies are provided, including therapeutic methods that utilizedosing regimens that differ from those utilized for non-chirallycontrolled compositions (e.g., of the same nucleotide sequence). In someembodiments, relatively larger individual doses of chirally controlled(e.g., chirally pure) oligonucleotides may be utilized as compared witha control (e.g., a stereorandom control). In some embodiments,relatively smaller individual doses of chirally controlled (e.g.,chirally pure) oligonucleotides may be utilized as compared with acontrol (e.g., a stereorandom control). In some embodiments, relativelyfewer doses of chirally controlled (e.g., chirally pure)oligonucleotides may be utilized as compared with a control (e.g., astereorandom control) within a given period of time.

Example 70: siRNA Agents Comprising Chirally Controlled OligonucleotidesInhibit PCSK-9

The present Example demonstrates successful inhibition of target geneexpression using siRNA agents comprised of chirally controlledoligonucleotides as described herein. Specifically, this Exampledescribes hybridization of individual oligonucleotide strands preparedthrough chirally controlled synthesis as described herein, so thatdouble-stranded chirally controlled siRNA oligonucleotide compositionsare provided. This Example further demonstrates successful transfectionof cells with such agents and, moreover, successful inhibition of targetgene expression.

siRNA Transfection of Chiral siRNA Molecules

Hep3B, or HeLa cells were reverse transfected at a density of 2.0×10⁴cells/well in 96-well plates. Transfection of siRNA was carried out withlipofectamine RNAiMax (Life Technologies, cat. No. 13778-150) using themanufacturer's protocol, except with a decreased amount of LipofectamineRNAiMax of 0.2 ul per well. Twelve, 1:3 siRNA duplex dilutions werecreated starting at 1 uM. 10 ul of 10×siRNA duplex was then lipoplexedwith a prepared mixture of 9.8 ul of serum-free medium and 0.2 ul ofLipofectamine RNAiMax per well. After a 10-15 minute incubation, 2.0×10⁴cells in 80 ul of EMEM cell growing media (ATCC, 30-2003) were added tobring the final volume to 100 ul per well. Two separate transfectionevents were performed for each dose.

24 hours after transfection Hep3B or HeLa cells were lysed and PCSK9mRNA was purified using MagMAX™-96 Total RNA Isolation Kit (LifeTechnologies, AM1830); 15 ul of cDNA was synthesized with High CapacitycDNA Reverse Transcription Kit with RNase Inhibitor (Life Technologies,4374967). Gene expression was evaluated by Real-Time PCR on aLightcycler 480 (Roche) using a Probes Master Mix (Roche, 04 707 494001) according to manufacturer's protocol using a FAM-labeled Taqmanprobe-set for PCSK9 (Life Technologies, Hs03037355_m1) and a VIC-labeledGAPDH primer-limited endogenous control (Life Technologies, NM_002046.3)

IC50s and Data Analysis

Delta delta Ct method was used to calculate values. Samples werenormalized to hGAPDH and calibrated to mock transfected and untreatedsamples. A stereo-random molecule was used as positive control. The dataare represented as a mean of 2 biological replicates using GraphpadPrism. A four-parameter linear regression curve was fitted to the dataand the bottom and top were constrained to a 0 and 100 constantsrespectively in order to calculate a relative IC50.

TABLE E-29 Relative IC50 values [Hep3B Transfection] LogIC50 HillSlopeBottom Top Mean SEM Mean SEM IC50 [nM] ONT-D1 0 100 −0.39 0.12 −0.270.02 0.41 ONT-D2 0 100 −0.85 0.14 −0.24 0.03 0.14 ONT-D3 0 100 −0.620.14 −0.24 0.02 0.24 ONT-D4 0 100 −0.49 0.09 −0.28 0.02 0.33 ONT-D5 0100 −0.32 0.09 −0.31 0.02 0.48

TABLE E-30 Relative IC50 values [HeLa Transfection] LogIC50 HillSlopeBottom Top Mean SEM Mean SEM IC50 ONT-D1 0 100 −1.73 0.07 −0.61 0.050.02 ONT-D2 0 100 −1.94 0.07 −0.66 0.07 0.01 ONT-D3 0 100 −1.72 0.08−0.62 0.07 0.02 ONT-D4 0 100 −1.26 0.06 −0.74 0.06 0.06 ONT-D5 0 100−1.03 0.08 −0.86 0.12 0.09

TABLE E-31 Relative IC50 values of siRNAs with 3 Phosporothioatestereo-centers[HeLa Transfection] LogIC50 HillSlope Bottom Top Mean SEMMean SEM IC50 ONT-D7 0 100 0.75 0.15 −0.24 0.02 5.60 ONT-D8 0 100 0.390.09 −0.32 0.02 2.45 ONT-D9 0 100 0.43 0.13 −0.22 0.02 2.68 ONT-D10 0100 1.57 0.20 −0.27 0.03 36.86 ONT-D11 0 100 2.92 0.33 −0.12 0.01 839.42ONT-D12 0 100 −0.17 0.10 −0.38 0.03 0.68 ONT-D13 0 100 1.20 0.21 −0.390.08 15.97 ONT-D14 0 100 −0.22 0.18 −0.43 0.07 0.60 ONT-D15 0 100 −0.250.16 −0.41 0.06 0.57

FIGS. 45-50 present results of these studies. FIG. 45 shows the % PCSK-9mRNA remaining after Hep3B treatment with siRNA duplex. FIG. 46 showsthe % PCSK-9 mRNA remaining after Hep3B treatment with siRNA duplexcurve fit. FIG. 47 shows the % PCSK-9 mRNA remaining after HeLatreatment with siRNA duplex. FIG. 48 shows the % PCSK-9 mRNA remainingafter HeLa treatment with siRNA duplex curve fit. FIG. 49 shows the %PCSK-9 mRNA remaining after HeLa treatment with siRNA duplex containing3 Phosphorothiate stereo-centers. FIG. 50 shows the % PCSK-9 mRNAremaining after HeLa treatment with siRNA duplex containing 3Phosphorothiate stereo-centers curve fit.

As can be seen, in molecules with a single stereochemically definedphosphorothioate in each strand, a slightly increased potency wasobserved with molecules with Sp stereo-chemistry at the phosphorothioatein both sense and antisense strands While the specifically exemplifiedsiRNA agents contained only one chiral center per strand, those skilledin the art, reading the present disclosure, will recognize thesignificance of the demonstrated differential effect as proof ofprinciple that presence of chirality can impact activity.

The impact of chirality is more pronounced in siRNAs with more than twostereo-centers. In siRNAs with 3 chiral phosphorothioates (one in thesense strand and two in the antisense strand), an Sp stereochemistry atthe 5′ end (between nucleotides n1 and n2) combined with a Rpstereochemistry at the 3′ end (between nucleotides n20 and n21) of theantisense strand had deleterious effect on potency. However, an Rpstereochemistry at the 5′ end (between nucleotides n1 and n2) combinedwith a Sp stereochemistry at the 3′ end (between nucleotides n20 andn21) of the antisense strand showed a significant PCSK-9 mRNA knockdownimprovement over stereo-random control siRNA. The results suggest thatboth strands are affected by the stereochemistry of thephosphorothioate. It is likely that the observed efficacy differentialwill increase for agents with larger numbers of chiral centers and,moreover that both location and type of chiral center can impactactivity, for example through effects on stability, potency, and/orboth. Those skilled in the art, reading the present disclosure, willtherefore appreciate that it provides teachings of both single anddouble-stranded chirally controlled oligonucleotide compositions, anduses thereof that, in at least some embodiments, distinguish suchchirally controlled oligonucleotides and agents from stereorandom agentsof identical sequence.

Example 71. Additional Exemplary Oligonucleotides and Synthesis Thereof

Oligonucleotides N101-N104 were synthesized using the automatedsynthesis on ABI-394 DNA/RNA synthesizer according to the syntheticcycle summarized on Table E-32, using 1 μmol synthesis column and 1.7μmol of oxalyl linked dG^(CE,Pac) on HCP. The synthesis cycle wasperformed with removal of the terminal 5′-O-DMTr group (DMT Off). Aftercompletion of the automated oligonucleotide synthesis, the HCP supportwas washed with dry ACN and dried under vacuum. The dry HCP was placedin a plastic vial and was treated with 1 mL of sat. NH₃/Py for 1 h at55° C. then 1 mL of dry propylamine in dry pyridine (in a 1:4 ratio) for18 h at RT. The solvents were then evaporated and the residue wasre-suspended with ˜pH 1.5 HCl aqueous solution containing 10% DMSO andthe HCP support was filtered off. The crude product was purified byreverse phase preparative HPLC (According to the procedure describedbelow). The fractions having purity above 95% were pooled, concentratedand desalted by reverse-phase HPLC (According to the procedure describedbelow). The final desalted product was lyophilized from water.

TABLE E-32 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI-394 Used for the Synthesis of oligonucleotidesN101-N102. delivery time step reaction reagent (sec) wait time (sec) 1detritylation 3% TCA in DCM 3 + 60 + 10 N.A. 2 coupling 0.15Mphosphoramidite in 5 + 4 30 + 600 ACN + 1.2M CMPT in ACN 3 capping 1 5%Pac₂O in THF/2,6- 20 60 lutidine 4 capping 2 5% Pac₂O in THF/2,6- 20 60lutidine + 16% NMI in THF 5 sulfurization 0.3M S-(N- 7 + 1.5 × 4 360 + 3× 180 + methylpiperazinoethyl- 900 ToluylThioSulfonate

Example 72. HPLC Method for N101 and N102

Buffer A: 50 mM TEAA, pH 9.6 (adjusted with TEA)

Buffer B: ACN Column: XBridge C₈, 3.5 μm, 4.6×50 mm, Part #186003034

Buffer heater set temperature=35° C.Signal monitored at 254 and 280 nmGradient used:

Time Flow (ml/min) % A % B Curve Initial 99 1 2 1 99 1 1 22 1 70 30 6 251 5 95 6 25.5 1 5 95 6 30 1 99 1 1

Example 73. General UPLC-LCMS Method for N101 and N102

Buffer A: 10 mM ammonium formate, pH 9.5 (adjusted with NH₃ aq.)

Buffer B: ACN Column: UPLC@OST C₁₈ 1.7 μm, 2.1×500 mm

Column temperature=35° C.Gradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 0.5 99 1 2 0.5 99 1 1 120.5 60 40 6 13 0.5 5 95 6 13.5 0.5 5 95 6 15 0.5 99 1 1

Example 74. General Purification Method for N101 and N102

Buffer A: 50 mM TEAA pH=9.6 (adjusted with TEA)

Buffer B: ACN

Column: XBridge Prep C₁₈, 5 μm, C₁₈, 250×10 mm, Part #186003256Buffer heater set temperature=RTSignal monitored at 254 and 280 nmGradient used:

Time Flow (ml/min) % A % B Curve Initial 99 1 5 4 99 1 1 10 4 95 5 6 304 74 26 6 35 4 5 95 6 36 4 5 95 6 38 4 99 1 6 45 4 99 1 1100 μL of 49-51% phosphoric acid was added to each fraction (pH 3) andchecked by HPLC, then stored at −80° C. until frozen. The fractions werekept on lypholizer for a couple of hours until ½ volumes then stored at−80° C.

Example 75. General Desalting Method for N101 and N102

Buffer A: 10 mM formic acid

Buffer B: ACN

Column: XBridge Prep C₁₈, 5 μm, C₁₈, 250×10 mm, Part #186003256Buffer heater set temperature=RTSignal monitored at 254 and 280 nmGradient used:

Time Flow (ml/min) % A % B Curve Initial 100 0 12 4 100 0 1 13 4 65 35 621 4 65 35 1 21.5 4 100 0 6 30 4 100 0 1Collected fractions were stored in −80° C. until frozen and kept onlypholizer until dryness and checked by HPLC.

Example 76. Synthesis of Oligonucleotide N101(All-Rp)-d[5mCs16As16Gs16T]

Oligonucleotide N101 was synthesized as described above and purified. RTin RP-HPLC: (HPLC method N1): 15.9 min. UPLC/ESI-MS: Calcd: 1614.64;Found [+H]: 1615.06.

Example 77. Synthesis of Oligonucleotide N102(All-(Sp)-d[5mCs16As16Gs16T])

Oligonucleotide N102 was synthesized as described above and purified. RTin RP-HPLC (HPLC method N1): 16.4 min. UPLC/ESI-MS: Calcd: 1614.64;Found [+H]: 1614.97.

Example 78. Synthesis of Oligonucleotide N103(All-Rp)-d[5mCs16As16Gs16Ts165mCs16Ts16Gs165mCs16Ts16Ts165mCs16G] (SEQID NO: 125))

Oligonucleotide N103 was synthesized as described above. RT in RP-HPLC(HPLC method N1): 18.4 min. UPLC/ESI-MS: Calcd for C₁₉₇H₃₁₁N₆₂O₆₂P₁₁S₁₁:5233.38; Found: 5234.7.

Example 79. Synthesis of Oligonucleotide N104(All-(Sp)-d[5mCs16As16Gs16Ts165mCs16Ts16Gs165mCs16Ts16Ts165mCs16G] (SEQID NO: 125))

Oligonucleotide N104 was synthesized as described above. RT in RP-HPLC(HPLC method N1): 18.7 min. UPLC/ESI-MS: Calcd: 5233.38; Found: 5232.9.

Oligonucleotides N105-N106 were synthesized using the automatedsynthesis on ABI-394 DNA/RNA synthesizer according to the syntheticcycle summarized on Table E-33, using 1 μmol synthesis column and 1.7μmol of oxalyl linked dG^(CE,Pac) on HCP. The synthesis cycle wasperformed with removal of the terminal 5′-O-DMTr group (DMT Off). Aftercompletion of the automated oligonucleotide synthesis, the HCP supportwas washed with dry ACN and dried under vacuum. The dry HCP was placedin a plastic vial and was treated with 1 mL of sat. NH₃/i-PrOH for 16 hat 55° C. The solvent was then evaporated and the residue wasre-suspended with pH 7.0 aqueous solution containing 10% DMSO and theHCP support was filtered off. The crude product is purified by reversephase preparative HPLC. The fractions having purity above 95% arepooled, concentrated and desalted by reverse-phase HPLC. The finaldesalted product is lyophilized from water.

TABLE E-33 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI-394 Used for the Synthesis of oligonucleotidesN105-N106. delivery time step reaction reagent (sec) wait time (sec) 1detritylation 3% TCA in DCM 3 + 60 + 10 N.A. 2 coupling 0.15Mphosphoramidite in 5 + 4 30 + 600 ACN + 1.2M CMPT in ACN 3 capping 1 5%Pac₂O in THF/2,6- 20 60 lutidine 4 capping 2 5% Pac₂O in THF/2,6- 20 60lutidine + 16% NMI in THF 5 sulfurization

7 + 1.5 × 4 360 + 3 × 180 + 900 

Example 80. HPLC Method for N101 and N102 Buffer A: 50 mM TEAA, pH 7.0Buffer B: ACN Column: XBridge C₈, 3.5 μm, 4.6×150 mm, Part #186003034

Buffer heater set temperature=35° C.Signal monitored at 254 and 280 nmGradient used:

Time Flow (ml/min) % A % B Curve Initial 99 1 2 1 99 1 1 22 1 80 20 6 251 5 95 6 25.5 1 5 95 6 30 1 99 1 1

Example 81. General UPLC-LCMS Method for N101 and N102

Buffer A: 10 mM ammonium formate, pH 7.0

Buffer B: ACN Column: UPLC@OST C₁₈ 1.7 μm, 2.1×500 mm

Column temperature=35° C.Gradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 0.5 99 1 2 0.5 99 1 1 120.5 70 30 6 13 0.5 5 95 6 13.5 0.5 5 95 6 15 0.5 99 1 1

Example 82. Synthesis of Oligonucleotide N105 (All-(Rp)-d[5mCs9As9Gs9T])

Oligonucleotide N105 was synthesized as described above. RT in RP-HPLC:(HPLC method N2): 14.1 min. UPLC/ESI-MS: Calcd: 1977.78; Found [−H]:1978.8.

Example 83. Synthesis of Oligonucleotide N106 (All-(Sp)-d[5mCs9As9Gs9T])

Oligonucleotide N106 was synthesized and purified as described above. RTin RP-HPLC (HPLC method N2): 14.7 min. UPLC/ESI-MS: Calcd: 1977.78;Found [−H]: 1977.37.

Oligonucleotides xxx were synthesized using the automated synthesis onABI-394 DNA/RNA synthesizer according to the synthetic cycle summarizedon Table E-33, using 1 μmol synthesis column and 1.7 μmol of oxalyllinked dG^(CE,Pac) on HCP. The synthesis cycle was performed withremoval of the terminal 5′-O-DMTr group (DMT Off). After completion ofthe automated oligonucleotide synthesis, the HCP support was washed withdry ACN and dried under vacuum. The dry HCP was placed in a plastic vialand was treated with 1 mL of sat. NH₃/i-PrOH for 16 h at 55° C. Thesolvent was then evaporated and the residue was re-suspended with pH 7.0aqueous solution containing 10% DMSO and the HCP support was filteredoff. The crude product is purified by reverse phase preparative HPLC.The fractions having purity above 95% are pooled, concentrated anddesalted by reverse-phase HPLC. The final desalted product islyophilized from water.

TABLE E-34 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI-394. delivery time step reaction reagent (sec) wait time(sec) 1 detritylation 3% TCA in DCM 3 + 60 + 10 N.A. 2 coupling 0.15Mphosphoramidite in 5 + 4 30 + 600 ACN + 1.2M CMPT in ACN 3 capping 1 5%Pac₂O in THF/2,6- 20 60 lutidine 4 capping 2 5% Pac₂O in THF/2,6- 20 60lutidine + 16% NMI in THF 5 sulfurization

10 + 4 × 2 300 + 3 × 150 + 600 

Example 84. General Purification Method Buffer A: 50 mM TEAA Buffer B:ACN

Column: XBridge Prep C₁₈, 5 μm, C₁₈, 250×10 mm, Part #186003256Buffer heater set temperature=rtSignal monitored at 254 and 280 nmGradient used:

Time Flow (ml/min) % A % B Curve Initial 99 1 5 4 99 1 1 10 4 95 5 6 304 74 26 6 35 4 5 95 6 36 4 5 95 6 38 4 99 1 6 45 4 99 1 1

HPLC Method 5:

Time Flow (ml/min) % A % B Curve Initial 80 20 2 1 80 20 1 22 1 45 55 625 1 5 95 6 25.5 1 5 95 6 26 1 85 20 6 30 1 85 20 1

ONT-60: Oligonucleotide 149All-(Rp)-d[5mCs8As8Gs8Ts85mCs8Ts8Gs85mCs8Ts8Ts85mCs8G] (SEQ ID NO: 125)

The oligonucleotide was synthesized as described above. RT in RP-HPLC(HPLC method 5): 15.80 min. UPLC/ESI-MS: Calcd: 6345.6; Found: 6340.7.

ONT-69: All-(Sp)-d[5mCs16As8Gs8Ts85mCs8Ts8Gs85mCs8Ts8Ts85mCs8G] (SEQ IDNO: 125)

The oligonucleotide was synthesized as described above. RT in RP-HPLC(HPLC method 5): 18.61 min. UPLC/ESI-MS: Calcd: 6345.6; Found: 6340.6.

Example 85

The P-modified blockmer and altmer oligonucleotides containing bothstereodefined phosphorothioate diester and stereodefined morpholinoethylphosphorothioate triester internucleotidic linkages were synthesized onan ABI-394 DNA/RNA synthesizer according to the cycle summarized inTable E-4 using 1 μmol synthesis column and 1.7 μmol of oxalyl linkeddG^(CE,Pac) on HCP. Either stereodefined P-modified phosphorothioatelinkage was introduced at predetermined positions within the sequence byperforming either sulfurization step (1) or (2). The synthesis cycle wasperformed with removal of the terminal 5′-O-DMTr group (DMT Off). Thesolid support was washed with dry ACN and dried under a flux of argon.The dry HCP was placed in a plastic vial and was treated with 1 mL ofdry propylamine in dry pyridine (in a 1:4 ratio) for a period of 18 h atr.t. The solvents were then evaporated and the residue was re-suspendedin DMSO and the HCP support was filtered off. The crude product waspurified by reverse phase preparative HPLC. The fractions having purityabove 95% were pooled, concentrated and desalted by reverse-phase HPLC(Gradient of 0 to 80% ACN). The final desalted product was lyophilizedfrom water.

TABLE E-4 Summary for Oligonucleotide Synthesis on a DNA/RNA SynthesizerABI-394 Used for the Synthesis of Example 85. delivery time stepreaction reagent (sec) wait time (sec) 1 detritylation 3% TCA in DCM 3 +60 + 10 N.A. 2 coupling 0.15M phosphoramidite in 5 + 4 30 + 600 ACN +1.2M CMPT in ACN 3 capping 1 5% Pac₂O in THF/2,6-lutidine 20 60 4capping 2 5% Pac₂O in THF/2,6-lutidine + 20 60 16% NMI in THF 5sulfurization 0.3M S-Morpholinoethyl p- 10 + 4 × 2 300 + 3 × 150 +Chlorophenyl thiosulfonate 600 

6 sulfurization 0.3M S-cyanoethyl 10 + 4 × 2 300 + 3 × 150 +Methylthiosulfonate 600 

Example 86. General Purification Method for Example 85

Buffer A: 20 mM Phosphate pH=6.0 (adjusted with phosphoric acid)

Buffer B: ACN

Column: XBridge Prep C₁₈, 5 μm, C₁₈, 250×10 mm, Part #186003256Buffer heater set temperature=50° C.Signal monitored at 254 and 280 nmGradient used:

Time Flow (ml/min) % A % B Curve Initial 99 1 5 4 99 1 1 10 4 77 23 6 604 70 30 6 65 4 20 80 6 70 4 20 80 6 71 4 99 1 6 80 4 99 1 1

HPLC Method 6

Buffer A: 20 mM Ammonium acetate, pH 6.0

Buffer B: ACN Column: XBridge C₈, 3.5 μm, 4.6×150 mm, Part #186003034

Buffer heater set temperature=60° C.Signal monitored at 254 and 280 nmGradient used:

Time Flow (ml/min) % A % B Curve Initial 90 10 2 1 90 10 1 22 1 75 25 625 1 5 95 6 25 1 5 95 6 25.5 1 90 10 6 30 1 90 10 1

ONT-71: All-(Rp)-d[5mCs1As1GsTs5mCsTsGs5mCsTsTs1S5mCs1G] (SEQ ID NO:125)

The oligonucleotide was synthesized as described above. RT in RP-HPLC(HPLC method 6): 9.39 min. UPLC/ESI-MS: Calcd: 4297.9; Found: 4295.3

ONT-72: All-(Sp)-d[5mCs1As1GsTs5mCsTsGs5mCsTsTs15mCs1G] (SEQ ID NO: 125)

The oligonucleotide was synthesized as described above. RT in RP-HPLC(HPLC method 6): 10.84 min. UPLC/ESI-MS: Calcd: 4297.9; Found: 4295.7

ONT-73: All-(Rp)-d[5mCs1AsGs1Ts5mCs1TsGs15mCsTs1Ts5mCs1G] (SEQ ID NO:125)

The oligonucleotide was synthesized as described above. RT in RP-HPLC(HPLC method 6): 13.54 min. UPLC/ESI-MS: Calcd: 4524.2; Found: 4522.6

ONT-74: All-(Sp)-d[5mCs1AsGs1Ts5mCs1TsGs15mCsTs1Ts5mCs1G] (SEQ ID NO:125)

The oligonucleotide was synthesized as described above. RT in RP-HPLC(HPLC method 6): 15.52 min. UPLC/ESI-MS: Calcd: 4524.2; Found: 4521.0

Prodrug oligonucleotide properties such as: nuclease resistance, tissueaccumulation, cell penetration, endosomal escape, immunostimulation,duration of action, pharmacokinetics, etc. are all modulated andinfluenced by the stereochemistry of the chiral phosphorothioatebackbone linkages.

RNA oligonucleotides containing 2′-OH, 2′-OMe, 2′-F, 2′-deoxy,internucleotidic phosphodiester or internucleotidic stereodefinedphosphorothioate diester or internucleotidic stereodefinedphosphorothioate triester (ProDrug) (either releasing a internucleotidicphosphodiester (PO) or a internucleotidic stereodefined phosphorothioatediester (PS)) linkages are synthesized on ABI 394 DNA/RNA synthesizeraccording to the cycles summarized in Table E-35, Table E-36, Table E-37and Table E-38, using a 10 μmol capacity synthesis column loaded with130 mg (4.9 μmol) of oxalyl linked 5′-O-DMTr-2′-deoxythymidine preparedas previously described. Prior to synthesis, a preliminary capping step(capping 2) is performed and the synthesis is terminated with removal ofthe terminal 5′-O-DMTr groups. The oxidation step is performed using acommercially available 5-6 M solution of tert-butyl hydroperoxide (TBHP)in decane which was then diluted with four parts dichloromethane. Thestereospecific sulfurization step for internucleotidic stereodefinedphosphorothioate diester linkage is performed using the 0.3 MS-cyanoethylmethylthiosulfonate reagent following the coupling of thecorresponding chiral phosphoramide and the two-step capping process(Table E-36). The stereospecific sulfurization step for internucleotidicstereodefined phosphorothioate triester releasing a stereodefinedinternucleotidic phosphodiester (PS) linkage is performed using the 0.3M S—(N-morpholinoethylthioester-ethyl)-para-nitro-toluylthiosulfonatereagent following the coupling of the corresponding chiral phosphoramideand the two-step capping process (Table E-37). The stereospecificsulfurization step for internucleotidic stereodefined phosphorothioatetriester releasing an internucleotidic phosphodiester (PO) linkage isperformed using the 0.3 M S—(N-morpholinoethyl)-toluylthiosulfonatereagent following the coupling of the corresponding chiral phosphoramideand the two-step capping process (Table E-38). For 2′-OH RNAnucleotides, 2′-O-base protecting groups are used, such as 2′-O-PivOM(Debart et al., Chem. Eur. J., 2008, 14, 9135) or 2′-O-TC (Dellinger etal., J. Am. Chem. Soc., 2011, 133, 11540). Ultra-mild (C-Ac, G-Pac,A-Pac) protecting groups are used for all of the nucleobases.

Once the automated oligonucleotide synthesis cycle is completed and thefinal 5′-O-DMTr group is removed, the synthesis column is taken off theDNA/RNA synthesizer and dried under vacuum. 10 mL solution of 0.5 M1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 0.25 MN,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in ACN is continuouslyadded to the support through the synthesis column for 1 min withoutstopping the flow using a syringe attached to one end of the synthesiscolumn. The support is then washed with anhydrous ACN and dried undervacuum. Then, the dried support is transferred into an empty screw-capplastic vial and treated with 10% n-PrNH₂ solution in anhydrous pyridine(1.5 mL) at room temperature for 12 h. After that, the solvents areevaporated to dryness and the residue including the solid support isdissolved in 2 mL of water/DMSO (50/50) at pH 5, the support is filteredoff and the filtrates are collected, immediately frozen and stored at−80° C. prior to purification.

TABLE E-35 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI 394 delivery wait step reaction reagent time (sec) time(sec) 1 detritylation 3% TCA in DCM 3 + 120 + 10 N. A. 0.15M 2 couplingphosphoramidite in 7 + 6 30 + 600 ACN + 2M CMPT in ACN 5% Pac₂O inTHF/2,6- 3 capping lutidine + 16% NMI in 10 20 THF 4 oxidation 1.1Mtert-butyl 20 110 hydroperoxide in 4:1 dichloromethane:decane

TABLE E-36 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI 394 step reaction reagent delivery time (sec) wait time(sec) 1 detritylation 3% TCA in DCM 3 + 120 + 10 N.A. 2 coupling 0.15Mchiral 8 + 6 30 + 900 (2′-OMe phosphoramidite in RNA) ACN + 2M CMPT in30 + 600 (DNA) ACN 3 capping 1 5% Pac₂O in THF/2,6- 30 60 lutidine 4capping 2 5% Pac₂O in THF/2,6- 30 60 lutidine + 16% NMI in THF 5sulfurization 0.3M S-(2-cyanoethyl) 15 + 3 × 4 120 + 3 × 60 + 300methylthiosulfonate

TABLE E-37 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI-394. delivery time step reaction reagent (sec) wait time(sec) 1 detritylation 3% TCA in DCM 3 + 60 + 10 N.A. 2 coupling 0.15Mphosphoramidite in 5 + 4 30 + 600 ACN + 1.2M CMPT in ACN 3 capping 1 5%Pac₂O in THF/2,6- 20 60 lutidine 4 capping 2 5% Pac₂O in THF/2,6- 20 60lutidine + 16% NMI in THF 5 sulfurization 0.3M S-(N- 10 + 4 × 2 300 + 3× 150 + morpholinothioester-ethyl- 600  p-nitro-toluylthiosulfonate

TABLE E-38 Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer ABI-394. delivery time step reaction reagent (sec) wait time(sec) 1 detritylation 3% TCA in DCM 3 + 60 + 10 N.A. 2 coupling 0.15Mphosphoramidite in 5 + 4 30 + 600 ACN + 1.2M CMPT in ACN 3 capping 1 5%Pac₂O in THF/2,6-lutidine 20 60 4 capping 2 5% Pac₂O inTHF/2,6-lutidine + 20 60 16% NMI in THF 5 sulfurization 0.3MS-Morpholinoethyl 10 + 4 × 2 300 + 3 × 150 + Toluylthiosulfonate 600

Synthesis of Oligonucleotide: (Rp)-uucuAGAccuGuuuuGcuudTs10dT (SEQ IDNO: 121)

Synthesis of Oligonucleotide ONT-107: (Sp)-uucuAGAccuGuuuuGcuudTs10dT(SEQ ID NO: 121)

Synthesis of Oligonucleotide ONT-108: (Rp)-AAGcAAAAcAGGUCuAGAAdTs10dT(SEQ ID NO: 122)

Synthesis of Oligonucleotide ONT-109: (Sp)-AAGcAAAAcAGGUCuAGAAdTs10dT(SEQ ID NO: 122)

Synthesis of Oligonucleotide: (Rp, Rp)-as1AGcAAAAcAGGUCuAGAAdTsdT (SEQID NO: 122)

Synthesis of Oligonucleotide: (Sp, Rp)-as1GcAAAAcAGGUCuAGAAdTsdT (SEQ IDNO: 123)

Synthesis of Oligonucleotide: (Sp, Sp)-as1GcAAAAcAGGUCuAGAAdTsdT (SEQ IDNO: 123)

Synthesis of Oligonucleotide: (Rp, Sp)-as1GcAAAAcAGGUCuAGAAdTsdT (SEQ IDNO: 123)

Synthesis of Oligonucleotide: (All(Sp))-us1ucus1AGAccs1uGus1uus1uGcuudTs10dT (SEQ ID NO: 121)

Synthesis of Oligonucleotide: (All(Rp))-As10AGcAAAAcAGGs1UCuAs1GAs1AdTs10dT (SEQ ID NO: 122)

RNA strand thermal annealing and preparation of siRNA duplexes. Each RNAstrand is mixed with its complementary RNA strand in equimolarconcentration of 10 μM in 1×PBS. Total 0.5 mL solution is prepared foreach duplex and the mixture is heated at 90° C. for 2 min and is allowedto cool down over the course of several hours. The mixtures are thenstored at 4° C.

Following the thermal RNA strand annealing step, all the possible siRNAduplex combinations are prepared by annealing any of the Sense strandswith any possible complementary strand of the Antisense strands.

All prepared siRNA duplexes are evaluated in vitro for their PCSK9gene-silencing properties, following transfection in HeLa cells or Hep3Bcells.

All prepared siRNA duplexes with ProDrug groups are evaluated in vitrofor their PCSK9 gene-silencing properties, following free uptake inHep3B cells, Huh-7 cells or human primary hepatocytes.

Different potencies are observed, modulated by the number, the positionand the stereo architecture of the chiral phosphorothioate backbonelinkages, combined with the layers of additional chemical modificationsand ProDrug groups explored.

siRNA properties such as: nuclease resistance, cell penetration,endosomal escape, duplex thermodynamic stability, tridemnsionalstructure of the duplex, affinity towards the various mechanistic stepsof enzyme interactions, affinity towards the target mRNA, specificoff-target effects, immunostimulation, duration of action,pharmacokinetics, etc. are all modulated and influenced by thestereochemistry of the chiral phosphorothioate backbone linkages as wellas the presence or the absence of the ProDrug group.

The attachment of PO and PS releasing ProDrug groups to the siRNAduplexes enhances their intracellular delivery and free uptake in theabsence of transfection reagent or targeting ligand.

Example 87: Stability Studies of Diastereomerically PureOligonucleotides

The present Example compares the in vitro stability of chirally pureoligonucleotides with that observed for the “parent” stereorandommixture (i.e., for a composition containing oligonucleotides of the samesequence as the chirally pure oligonucleotides but not displaying chiralpurity, for example as a result of having been prepared via astereorandom process). Seven chirally pure oligonucleotides, each ofwhich had a sequence complementary to that of a particular targettranscript or gene encoding a protein of interest were synthesized,formulated, and assessed for metabolic stability using three differentbiological matrices: snake venom phosphodiesterase (svPDE), nuclease P1(nP1) and rat whole liver homogenates. Levels of full-lengtholigonucleotide were quantified by IEX-HPLC after different incubationperiods. The results presented in this Example demonstrate, forinstance, that chirally pure oligonucleotide compositions can havesignificantly different metabolic stability as compared with anappropriate reference (e.g., a preparation of oligonucleotides of thesame sequence but different chiral specificity, including particularlystereorandom preparations), and specifically as compared with a“parental” stereorandom preparation. In this Example, oligonucleotideshaving a sequence antisense to (and therefore targeting) humanApolipoprotein-B (ApoB) were used for proof-of-concept in metabolicstability studies.

Snake Venom Phosphodiesterase (svPDE) Digestion Study forOligonucleotides ONT-75, ONT-77, ONT-80, ONT-81, ONT-87, ONT-88, ONT-89and ONT-41

We used the protocol reported by Oka et al. (J. Am. Chem. Soc. 2008,130, 16031-16037) with minor modifications. Purified oligonucleotide (10nmoles) in water (50 μL) was added to the aqueous solution (450 μL, pH8.6) containing svPDE (4×10⁻³ units), 100 mM Tris-HCl and 15 mM MgCl₂.The mixture was incubated at 37° C. with shaking at 400 rpm. A 50 μLaliquot was taken at each time point (0 h, 12 h, 1 d, 2 d, 3 d, 4 d, 5d, 6 d and 7 d) and was quenched with 25 μL of 150 mM EDTA, 2 μL ofProteinase K solution (20 mg/mL) and 30 μL of Lysis buffer (Qiagen,#129115) and the mixture was heated at 60° C. for 20 min. 5 μL ofinternal standard (5′-GCGTTTGCTCTTCTTCTTGCGTTTTTT-3′ (SEQ ID NO: 126), a27-mer oligonucleotide (underlined nucleotides are 2′-MOE modified),(200 μM) was added to the aliquot. Quantification analyses wereperformed by IEX-HPLC and metabolite identification was carried out byUPLC/MS. The results were illustrated in FIG. 59.

IEX-HPLC analysis showed degradation of phosphorothioate 20-mer duringincubation with svPDE with no significant difference betweenstereoisomers. LCMS analysis of metabolites revealed that the majorityof the degradation products were formed as a result of desulfurization.As reported previously by Prakash et al. (Biochemistry 2002, 41,11642-11648), Prhavc et al. (Org. Lett., 2003, 5, 2017-2020) and others,we also observed that 2′-MOE modifications at 5′ and 3′-ends protectthese oligomers from svPDE digestion which is a 3′-5′ exonuclease.

Nuclease P1 (nP1) Digestion Study for Oligonucleotides ONT-75, ONT-77,ONT-80, ONT-81, ONT-87, ONT-88, ONT-89 and ONT-41

We employed the protocol reported by Oka et al. (J. Am. Chem. Soc. 2008,130, 16031-16037) with minor modifications. Purified oligonucleotide (10nmoles) in Water (50 μL) was added to an aqueous solution (500 μL, pH7.2) containing nP1 (20 units), 100 mM Tris-HCl and 1 mM ZnCl₂. Themixture was incubated at 37° C. with shaking at 400 rpm. A 50 μL aliquotwas taken at each time point (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 1 d, 2 d)and was quenched with 25 μL of stop buffer (150 mM EDTA, 2 μL ofProteinase K solution (20 mg/mL) and 30 μL of Lysis buffer (Qiagen,#129115) and the mixture was heated at 60° C. for 20 min. 5 μL ofinternal standard (5′-GCGTTTGCTCTTCTTCTTGCGTTTTTT-3′ (SEQ ID NO: 126)),a 27-mer oligonucleotide (underlined nucleotides are 2′-MOE modified)(200 μM) was added to the aliquot. Quantification analyses wereperformed by IEX-HPLC and metabolite identification was carried out byUPLC/MS. The results were illustrated in FIGS. 60-67.

Nuclease nP1 has previously been reported to specifically cleave DNAphosphorothioate linkages having an (Sp) absolute configuration at thephosphorus atoms (Porter et al., Biochemistry, 1983, 22, 1369-1377; Okaet al., J. Am. Chem. Soc., 2008, 130, 16031-16037). We observed asimilar pattern of cleavage for the 5-10-5 2′-MOE gapmerphosphorothioate oligonucleotides studied, where nP1 was found to digestefficiently the DNA core of the gapmer oligonucleotides at the (Sp)phosphorothioate centers, without affecting the 2′-MOE wings, which werestable independent of stereochemistry. The complete digestion of the DNAcore was observed in one hour for the stereorandom diastereomixtureoligonucleotide ONT-41, as well as for the stereopure oligonucleotidescontaining (Sp)-phosphorothioate internucleotidic linkages in the DNAcore (ONT-77, ONT-80, ONT-87, ONT-88 and ONT-89). All products ofcleavage were clearly identified by UPLC/MS. As previously reported inthe literature, we found that (Rp) phosphorothioate DNA was clearly nota substrate for nP1. The two chirally pure oligonucleotides having (Rp)phosphorothioate DNA cores (ONT-75 and ONT-81) were completely stable tonP1 for an incubation period of 1 h at 37° C. ONT-75 and ONT-81 showedc.a. 10-15% loss of the full length products during the course ofincubation over several days. The results presented in this Exampledemonstrate, for instance, that chirally pure oligonucleotidecompositions can have significantly different metabolic stability in annP1 assay as compared with an appropriate reference (e.g., a preparationof oligonucleotides of the same sequence but different chiralspecificity, including particularly stereorandom preparations), andspecifically as compared with a “parental” stereorandom preparation.

General IEX-HPLC Method for the Analysis of Enzyme Digestion Products

Buffer A: 10 mM TrisHCl, 50% ACN, pH 8.0 Buffer B: 10 mM TrisHCl, 800 mMNaClO₄, 50% ACN, pH 8.0 Column: DIONEX, DNAPac, PA-100, 4.0×250 mm, Part#063000

Column temperature=60° C.Signal monitored at 254 and 280 nmGradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 95 5 2 1 95 5 1 3 1 75 256 10 1 35 65 6 10.1 1 95 5 6 12.5 1 95 5 1

General UPLC-LCMS Method for the Analysis of Enzyme Digestion Products.

Buffer A: 15 mM TEA, 400 mM HFIP, Water Buffer B: 50:50 BufferA/Methanol Column: UPLC@OST C₁₈ 1.7 μm, 2.1×500 mm

Column temperature=60° C.Gradient used:

Time (min) Flow (mL/min) % A % B Curve Initial 0.2 70 30 2 0.2 70 30 127 0.2 35 65 6 27.5 0.2 5 95 6 28.5 0.2 5 95 6 29 0.2 70 30 6 30 0.2 7030 1

In Vitro Metabolic Stabilities of Human Diastereomerically PureOligonucleotides in Preincubated Rat Whole Liver Homogenates

The metabolic stability of chirally pure oligonucleotides in in vitrorat whole liver homogenates was measured. The protocol employed here haspreviously been reported and used to evaluate the stability ofoligonucleotide drugs.

Protocol: The protocol reported by Geary et al was employed in thecurrent study (Oligonucleotides, 2010, 20, 309) with some modifications.

Test system: Two male Sprague-Dawley rats (Rattus norvegicus) weresupplied by Charles River Laboratories, Inc., (Hollister, Calif.).

Tissue Collection: Animals were acclimated to the study room for twodays prior to tissue collection. At the time of tissue collection,animals were anesthetized with an intraperitoneal (IP) injection ofsodium pentobarbital solution. Liver perfusion was performed using 500mL of chilled saline/animal, administered via the hepatic portal vein.After perfusion, the livers were dissected and maintained on ice. Liverswere minced into small pieces then weighed.

Liver Homogenate Preparation: The minced pieces of liver tissues weretransferred to tared 50 mL centrifuge tubes and weighed. Chilledhomogenization buffer (100 mM Tris pH 8.0, 1 mM magnesium acetate, withantibiotic-antimycotic agents) was added to each tube, such that thetube(s) contained 5 mL of buffer per gram of tissue. Using a QIAGENTissueRuptor tissue homogenizer, the liver/buffer mixture washomogenized while maintaining the tube on ice. The protein concentrationof the liver homogenate was determined using a Pierce BCA protein assay.Liver homogenates were divided into 1 mL aliquots, transferred tocryovials and stored at −60° C.

Incubation Conditions: 1 mL aliquots of frozen liver homogenate (proteinconcentration=31.9 mg/mL) were thawed and incubated at 37° C. for 24 h.Six eppendorf tubes (2 mL) were taken and 450 μL of homogenate was addedin each tube. 50 μL of test oligonucleotide (200 μM) was added to eachtube. Immediately after mixing, 125 μL of (5×) stop buffer (2.5% IGEPAL,0.5 M NaCl, 5 mM EDTA, 50 mM Tris, pH=8.0) and 12.5 μL of 20 mg/mLProteinase K (Ambion, # AM2546) was added to one tube for a 0 hour timepoint. The mixture was then heated at 60° C. for one hour. The remainingreaction mixtures were incubated at 37° C. with shaking at 400 rpm on aVWR Incubating Microplate shaker. After incubation for a designatedperiod (1, 2, 3, 4, and 5 days), each mixture was treated with 125 μL of(5×) stop buffer (2.5% IGEPAL, 0.5 M NaCl, 5 mM EDTA, 50 mM Tris,pH=8.0) and 12.5 μL of 20 mg/mL Proteinase K (Ambion, # AM2546).

Work up and Bioanalysis: (5′-GCGTTTGCTCTTCTTCTTGCGTTTTTT-3′ (SEQ ID NO:126)), a 27-mer oligonucleotide (underlined nucleotides are 2′-MOEmodified) was used as the internal standard for quantitation ofdiastereomerically pure oligonucleotides. 50 μL of internal standard(200 μM) was added to each tube followed by addition of 250 μL of 30%ammonium hydroxide, 800 μL of Phenol:Chloroform:isoamyl alcohol(25:24:1). After mixing and centrifugation at 600 rpm, the aqueous layerwas evaporated on speed vac to 100 μL and loaded on a Sep Pak column(C18, 1 g, WAT 036905). All the aqueous washings of the Sep pak columnwere tested with quick IEX-HPLC method to ensure that no product wasfound there. The acetonitrile eluate was concentrated to dryness anddissolved in 100 μL water and analyzed using RP-HPLC.

Eluant A=10 mM Tributylammonium acetate, pH=7.0

Eluant B=ACN

Column: XTerra MS C₁₈, 3.5 μm, 4.6×150 mm, Part number: 186000432

Column Temperature=60° C. HPLC Gradient:

Time Flow % A % B Curve 1 1.0 65 35 2 5.0 1.0 65 35 1 3 30.0 1.0 40 60 64 35.0 1.0 5 90 6 5 36.0 1.0 65 35 6 6 40.0 1.0 65 35 1

FIG. 68 shows that different chirally pure oligonucleotides havedifferent metabolic stability profiles in rat whole liver homogenate.The experiment also demonstrates that the diastereoisomeric mixtureONT-41 has a different metabolic stability profile compared withdiastereomerically pure stereodefined oligonucleotides with the samesequence and chemical composition (ONT-75 and ONT-77 are used here asexamples, but this observation, as appreciated by a person of ordinaryskill in the art, may be extrapolated to other possiblediastereomerically pure stereodefined phosphorothioate diastereoisomersof this molecule).

The oligonucleotide ONT-75, having a stereocontrolled phosphorothioatebackbone with full Rp absolute configuration at all phosphorus atoms wasdegraded completely in one day in preincubated rat whole liverhomogenates at 37° C., demonstrating the lowest metabolic stability ofthe three used in the study.

The other diastereomerically pure stereodefined phosphorothioateoligonucleotide ONT-77 (having absolute configuration: 5R-10S-4R) wasmore than two-fold more stable than the stereorandom ONT-41(Mipomersen). After incubation for 5 days 40% of full lengtholigonucleotide remained for the diastereomerically pure ONT-77 vs.approximately 15% of full length oligonucleotide for the stereorandomONT-41 (Mipomersen). A direct comparison between ONT-77 and ONT-41clearly demonstrated a significantly higher metabolic stability for thestereocontrolled isomer 5R-10S-4R (ONT-77), throughout all of theanalyzed time points. The stereodefined architecture of thediastereomerically pure isomer ONT-77 clearly affects the rate ofdegradation and the overall metabolic stability of this oligonucleotide.These observations lead to the conclusion, without the intention to belimited by theory, that the Sp stereochemistry applied to the DNA coreof the 5-10-5 gapmer oligonucleotide provides enhanced endonucleaseresistance and hence enhanced metabolic stability compared with thestereorandom diastereomixture. Some other stereo architectures used forother diastereomerically pure stereocontrolled phosphorothioate isomersof this sequence would be expected to show even higher metabolicstability in this experiment.

While not wishing to be limited by theory, the results presented in thisExample demonstrate, for instance, that Rp stereochemistry at thephosphorothioate linkage (ONT-75) is less metabolically stable in ratliver homogenates than ONT-77, which contains the Sp DNA core. Theresults presented in this Example also demonstrate, for instance, thatchirally pure oligonucleotide compositions can have significantlydifferent metabolic stability in rat liver homogenate as compared withan appropriate reference (e.g., a preparation of oligonucleotides of thesame sequence but different chiral specificity, including particularlystereorandom preparations), and specifically as compared with a“parental” stereorandom preparation. In this Example, oligonucleotideshaving a sequence antisense to (and therefore targeting) humanApolipoprotein-B (ApoB) were used for proof-of-concept in metabolicstability studies. A provided chirally controlled oligonucleotides mayhave increased or decreased stability towards endogenous enzymes. Byproviding chirally controlled oligonucleotide and compositions andmethods thereof, the present invention provided oligonucleotides andcompositions that have enhanced pharmacological properties than knownchirally uncontrolled oligonucleotides and their compositions.

In Vitro Metabolic Stabilities of Human PCSK9 siRNA Duplexes HavingStereocontrolled Phosphorothioate Diester Linkages in Human Serum.

A protocol similar to previously reported procedures (Oka et al., J. Am.Chem. Soc. 2008, 130, 16031-16037) is used. siRNA duplexes (2.5 μmoles)in water (50 μL) are added to human serum (450 μL). The mixture isincubated at 37° C. with shaking at 400 rpm. 50 μL aliquots are takenout at each time point (0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h)and are quenched with 25 μL of 150 mM EDTA, 2 μL of Proteinase Ksolution (20 mg/mL) and 30 μL of Lysis buffer (Qiagen, #129115) and themixture is heated at 60° C. for 20 min. 5 μL of Internal standard(5′-GCGTTTGCTCTTCTTCTTGCGTTTTTT-3′ (SEQ ID NO: 126), a 27-meroligonucleotide (underlined nucleotides are 2′-MOE modified) (200 μM) isadded to the aliquot. Quantification analyses are performed by IEX-HPLCand metabolite identification is carried out by UPLC/MS.

In some embodiments, the siRNA duplexes having a 3′ terminaldiastereomerically pure phosphorothioate with the Sp configuration showhigher metabolic stability in human serum compared to the siRNAs havingthe phosphorothioate with the Rp configuration, or the stereorandomdiastereomixture, at the same position. In other embodiments, the siRNAduplexes having a 5′ terminal diastereomerically pure phosphorothioatewith the Sp configuration show higher metabolic stability in human serumcompared to the siRNAs having the phosphorothioate with the Rpconfiguration, or the stereorandom diastereomixture, at the sameposition. In other embodiments, siRNA duplexes having diastereomericallypure phosphorothioates with the Sp configuration at both the 3′ and the5′ extremities show higher metabolic stability in human serum comparedto the siRNAs having the phosphorothioate with the Rp configuration, orthe stereorandom diastereomixtures, at the same positions. In otherembodiments, siRNA duplexes having multiple diastereomerically purephosphorothioates with the Sp configuration show higher metabolicstability in human serum compared to the siRNAs having multiplephosphorothioates with the Rp configuration, or the stereorandomdiastereomixtures. In other embodiments, siRNA duplexes having fullbackbone of diastereomerically pure phosphorothioates with the Spconfiguration show higher metabolic stability in human serum compared tothe siRNAs having the full backbone of phosphorothioates with the Rpconfiguration, or the stereorandom diastereomixtures.

Example 88. Chirally Controlled Oligonucleotide Compositions ShowDifferent Potency In Vitro as Compared with Chirally UncontrolledCompositions Having the Same Sequence

The present Example compares in vitro pharmacological activity ofchirally pure oligonucleotides with that observed for the “parent”stereorandom mixture (i.e., for a composition containingoligonucleotides of the same sequence as the chirally pureoligonucleotides but not displaying chiral purity, for example as aresult of having been prepared via a stereorandom process). Fourchirally pure oligonucleotides, each of which had a sequencecomplementary to that of a particular target transcript or gene encodinga protein of interest were synthesized, formulated, and transfected intoprimary mouse hepatocytes. mRNA levels were quantified to assess levelof suppression. In this Example, oligonucleotides having a sequenceantisense to (and therefore targeting) human Apolipoprotein-B (ApoB)were used for proof-of-concept in transgenic mice expressing human ApoB.

Transfection of Mouse Primary Hepatocytes with Oligonucleotides

C57BL6 male mice, 7 weeks of age were used to extract and plate mouseprimary hepatocytes in 96-well plates (without overlay)(Celsis/Bioreclamation IVT). Transfection of primary hepatocytes wascarried out with lipofectin (Life Technologies, cat. No. 18292-037)using the manufacturer's protocol, using 0.5 ul of Lipofectin per96-plate well. Twelve, 1:3 siRNA duplex dilutions were created startingat 6 uM. 10 ul of 10× oligo was then lipoplexed with a prepared mixtureof 9.5 ul of In vitro Gro HI Medium (Celsis Cat. Z99009) serum-freemedium and 0.5 ul of Lipofectin per well. After a 10-15 minuteincubation, 20 ul of lipoplexed oligo was added to primary hepatocytesin 80 ul of In vitro Gro HI Medium to bring the final volume to 100 ulper well. 24 hours after transfection, cells were lysed.

Apolipoprotein B mRNA Assay

Total mRNA was purified from cell lysates using MagMAX™-96 Total RNAIsolation Kit (Life Technologies, AM1830); 15 ul of cDNA was synthesizedwith High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor(Life Technologies, 4374967). Gene expression was evaluated by Real-TimePCR on a Lightcycler 480 (Roche) using a Probes Master Mix (Roche, 04707 494 001) according to manufacturer's protocol using the followingprimers: Mouse apolipoprotein B primers:

[Template: (GenBank Accession Number M35186)]

forward primer: (SEQ ID NO: 127) CGTGGGCTCCAGCATTCTA reverse primer:(SEQ ID NO: 128) AGTCATTTCTGCCTTTGCGTC PCR probe: (SEQ ID NO: 129)FAM-CCAATGGTCGGGCACTGCTCAA-TAMRA

Mouse GAPDH Primers:

forward primer: (SEQ ID NO: 130) GGCAAATTCAACGGCACAGT reverse primer:(SEQ ID NO: 131) GGGTCTCGCTCCTGGAAGAT PCR probe: (SEQ ID NO: 132)5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′

Data Analysis

The delta delta Ct method was used to calculate values. For each sample,the mean ApoB signal was normalized to the mean GAPDH signal, and thennormalized to the mean corresponding ratio for mock transfected anduntreated samples, to obtain the relative level of ApoB proteinexpression. The “parent” stereorandom mixture was used for reference. Afour-parameter linear regression curve was fitted to the data, and thebottom and top were constrained to a 0% and 100% constants respectivelyin order to calculate a relative IC50 using Graphpad Prism.

The in vitro dose-responses (FIGS. 71 and 72) show that the chirallypure oligonucleotides have significantly different potencies, withONT-83 being the most potent, and ONT-84 and ONT-85 being the leastpotent, with a 8.6-fold difference in IC50s (Table E-39).

TABLE E-39 IC50 values of Stereoisomers (ONT-83, -84, -85 or -86) forSuppression of Mouse Apolipoprotein B/GAPDH mRNA Levels in Primary MouseHepatocytes Bottom Top LogIC50 Hill Slope IC50 ONT-83 0 100 1.6 −0.435.8 ONT-82 0 100 1.8 −0.3 64.4 ONT-85 0 100 2.5 −0.3 308.0 ONT-84 0 1002.5 −0.8 307.8 ONT-86 0 100 1.7 −0.6 51.2

The results presented in this Example demonstrate, for instance, thatchirally pure oligonucleotide compositions can have significantlydifferent pharmacological activity in vitro as compared with anappropriate reference (e.g., a preparation of oligonucleotides of thesame sequence but different chiral specificity, including particularlystereorandom preparations), and specifically as compared with a“parental” stereorandom preparation. Those skilled in the art, in lightof this demonstration, will appreciate that chirally controlledoligonucleotide compositions provided by the present disclosure haveunexpected activities and characteristics.

EQUIVALENTS

Having described some illustrative embodiments of the invention, itshould be apparent to those skilled in the art that the foregoing ismerely illustrative and not limiting, having been presented by way ofexample only. Numerous modifications and other illustrative embodimentsare within the scope of one of ordinary skill in the art and arecontemplated as falling within the scope of the invention. Inparticular, although many of the examples presented herein involvespecific combinations of method acts or system elements, it should beunderstood that those acts and those elements may be combined in otherways to accomplish the same objectives. Acts, elements, and featuresdiscussed only in connection with one embodiment are not intended to beexcluded from a similar role in other embodiments. Further, for the oneor more means-plus-function limitations recited in the following claims,the means are not intended to be limited to the means disclosed hereinfor performing the recited function, but are intended to cover in scopeany means, known now or later developed, for performing the recitedfunction.

Use of ordinal terms such as “first”, “second”, “third”, etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements. Similarly, use of a), b), etc.,or i), ii), etc. does not by itself connote any priority, precedence, ororder of steps in the claims. Similarly, the use of these terms in thespecification does not by itself connote any required priority,precedence, or order.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

APPENDIX (A)

SEQ Gene ID Accession # Unigene # Symbol Name Sequence NO: NM_003467Hs.421986 CXCR4 Chemokine (C-X-C motif) 5′-UAAAAUCUUCCUGCCCACCdTdT-3′139 receptor 4 NM_003467 Hs.421986 CXCR4 Chemokine (C-X-C motif)5′-GGAAGCUGUUGGCUGAAAAdTdT-3′ 140 receptor 4 NM_006799.2 Hs.72026 PRSS21Protease, serine, 21 (testisin) 5′-CACAUCCAGCCCAUCUGUC(dTdT)-3′ 141NM_000117.1 Hs.522823 EMD Emerin 5′-CCGUGCUCCUGGGGCUGGG(dTdT)-3′ 142NM_001350.3 Hs.336916 DAXX Death-associated protein 65′-GGAGUUGGAUCUCUCAGAA(dTdT)-3′ 143 NM_003014.2 Hs.105700 SFRP4Secreted frizzled-related 5′-AAGTCCCGCTCATTACAAATT-3′ 144 protein 4NM_015062.3 Hs.533551 PPRC1 Peroxisome proliferative5′-AAGACCAGCCUCUUUGCCCAG-3′ 145 activated receptor, gamma,coactivator-related 1 NM_001005360.1 Hs.211463 DNM2 Dynamin 25′-GGACCAGGCAGAAAACGAG-3 146 NM_001904.2 Hs.476018 CTNNB1Catenin (cadherin-associated 5′-CUAUCAGGAUGACGCGG-3′ 147Protein), beta 1, 88 kDa NM_153831.2 Hs.395482 PTK2PTK2 protein tyrosine kinase 2 5′-AACCACCUGGGCCAGUAUUAU-dTT-3′ 148NM_001429.2 Hs.517517 EP300 E1A binding protein p3005′-UGACACAGGCAGGCUUGACUU-3′ 149 NM_005904.2 Hs.465087 SMAD7SMAD, mothers against DPP 5′ AA GCU CAA UUC GGA CAA CAA 150homolog 7 (Drosophila) G 3′ NM_001904.2 Hs.476018 CTNNB1Catenin (cadherin-associated 5′ AAG UCC UGU AUG AGU GGG 151protein), beta 1, 88 kDa AAC 3′ NM_175847.1 Hs.172550 PTBP1Polypyrimidine tract binding 5′-TCG ACG AAC ATC TAC AAC GCC 152protein 1 TGC TTC AAG AGA GCA GGC GTT GTA GAT GTT CTT TTT TT-3′NM_175847.1 Hs.172550 PTBP1 Polypyrimidine tract binding5′-TCG ACC AAT GAC AAG AGC CGT 153 Protein 1 GAC TTC AAG AGA GTC ACG GCTCTT GTC ATT GTT TTT TT-3′ NM_002659.2 Hs.466871 PLAURPlasminogen activator, 5′-GGTGAAGAAGGGCGTCCAA-3′ 154 urokinase receptorNM_033360.2 Hs.505033 KRAS2 V-Ki-ras2 Kirsten rat sarcoma5′GATCCGTTGGAGCTGTTGGCGTAGTTCA 155 2 viral oncogene homologAGAGACTACGCCAACAGCTCCAACTTTTTG GAAA-3′ NM_002959.4 Hs.485195 SORT1Sortilin 1 5′-AGGTGGTGTTAACAGCAGAG-3′ 156 NM_002959.4 Hs.485195 SORT1Sortilin 1 5′-AATGTTCCAATGCCCCACTC-3′ 157 NM_000743.2 Hs.89605 CHRNA3Cholinergic receptor, nicotinic, 5′-AACUGCCAGUGGCCAGGGCCU-3′ 158alpha polypeptide 3 NM_004859.2 Hs.491351 CLTCClathrin, heavy polypeptide 5′-AACCUGCGGUCUGGAGUCAAC-3′ 159 (Hc)NM_004859.2 Hs.491351 CLTC Clathrin, heavy polypeptide5′-UAAUCCAAUUCGAAGACCAAU-3′ 160 (Hc) NM_000038.3 Hs.158932 APCAdenomatosis polyposis coli 5′-AGGGGCAGCAACTGATGAAAA-3′ 161 NM_004850.3Hs.58617 ROCK2 Rho-associated, coiled-coil 5′-AAGGCATCGCAGAAGGTTTAT-3′162 containing protein kinase 2 NM_001274.2 Hs.24529 CHEK1CHKl checkpoint homolog (S. 5′-UCGAAGUACUCAGCGUAAG-3′ 163 pombe)NM_007194.3 Hs.291363 CHEK2 CHK2 checkpoint homolog (S.5′-GAACCUGAGGACCAAGAAC-3′ 164 pombe) NM_001901.1 Hs.410037 CTGFConnective tissue growth factor 5′-AATGTTCTCTTCCAGGTCAGCCCTGT 165 CTC-3′NM_001619.2 Hs.83636 ADRBK1 Adrenergic, beta, receptor5′-AAGAAGUACGAGAAGCUGGAG-3′ 166 kinase 1 NM_005160.2 Hs.517493 ADRBK2Adrenergic, beta, receptor 5′-AAGCAAGCUGUAGAACACGUA-3′ 167 kinase 2NM_005308.2 Hs.524625 GRK5 G protein-coupled receptor5′-AAGCCGUGCAAAGAACUCUUU-3′ 168 kinase 5 NM_001004106.1 Hs.235116 GRK6G protein-coupled receptor 5-AACAGUAGGUUUGUAGUGAGC-3′ 169 kinase 6NM_017556.1 Hs.530101 FBLP-1 Filamin-binding LIM protein-15′-AAAGGGGCAUCCACAGACAUC-3′ 170 NM_005857.2 Hs.132642 ZMPSTE24Zinc metallopeptidase (STE24 5′-TTATTCTTCTCTTTGGAGGA-3′ 171homolog, yeast) NM_005572 Hs.491359 LMNA Lamin A/C5′-ACTGGACTTCCAGAAGAAC-3′ 172 NM_015878.3 Hs.459106 OAZINOrnithine decarboxylase 5′-AATTGCACGTAATCACCCAAA-3′ 173antizyme inhibitor NM_015878.3 Hs.459106 OAZIN Ornithine decarboxylase5′-AAGAAATACAAGGAAGATGAG-3′ 174 antizyme inhibitor NM_001664.2 Hs.247077RHOA Ras homolog gene family, 5′-GACAUGCUUGCUCAUAGUCTT-3′ 175 member ANM_175744.3 Hs.502659 RHOC Ras homolog gene family,5′-GACCUGCCUCCUCAUCGUCTT-3′ 176 member C NM_000041.2 Hs.515465 APOEApolipoprotein E 5′-AAGGTGGAGCAAGCGGTGGAG-3′ 177 NM_000041.2 Hs.515465APOE Apolipoprotein E 5′-AAGGAGTTGAAGGCCTACAAA-3′ 178 AF520590.1Hs.53600 BAK1 BCL2-antagonist/killer 1 5′-UGCCUACGAACUCUUCACCdTdT-3′ 179NM_138761.2 Hs.159428 BAX BCL2-associated X protein5′-UAUGGAGCUGCAGAGGAUGdTdT-3′ 180 NM_005733.1 Hs.73625 KIF20AKinesin family member 20A 5′-TTGGCCAAGCCACACACAG-3′ 181 NM_005733.1Hs.73625 KIF20A Kinesin family member 20A 5′-GTTCTCAGCCATTGCTAGC-3′ 182NM_005733.1 Hs.73625 KIF20A Kinesin family member 20A5′-GGCAGCATGTATTGCTGAG-3′ 183 NM_014034.1 Hs.292316 ASF1AASF1 anti-silencing function 1 5′-AAUCCAGGACUCAUUCCAGAU-3′ 184homolog A (S. cerevisiae) NM_014034.1 Hs.292316 ASF1AASF1 anti-silencing function 1 5′-AAGUGAAGAAUACGAUCAAGU-3′ 185homolog A (S. cerevisiae) NM_018154.1 Hs.26516 ASF1BASF1 anti-silencing function 1 5′-AACAACGAGUACCUCAACCCU-3′ 186homolog B (S. cerevisiae) NM_022110.3 Hs.520042 WISp39FK506 binding protein like 5′ AACGCUUGAGCUGGAAGG 3′ 187 NM_022110.3Hs.520042 WISp39 FK506 binding protein like 5′-CCUUCAAGCUUCUGAUCUC-3′188 NM_000389.2 Hs.370771 CDKN1A Cyclin-dependent kinase5′-AACUUCGACUUUGUCACCGAG-3′ 189 inhibitor 1A (p21, Cip1) NM_004064.2Hs.238990 CDKN1B Cyclin-dependent kinase 5′-AAGCACUGCAGAGACAUGGAAG-3′190 inhibitor 1B (p27, Kip1) NM_033084.2 Hs.208388 FANCD2Fanconi anemia, 5′-AACAGCCATGGATACACTTGA-3′ 191 complementation group D2NM_001641.2 Hs.73722 APEX1 APEX nuclease 5′-AATGACAAAGAGGCAGCAGG-3′ 192(multifunctional DNA repair  enzyme) 1 NM_001641.2 Hs.73722 APEX1APEX nuclease 5′-AACCTGCCACACTCAAGATC-3′ 193 (multifunctional DNA repairenzyme) 1 NM_001641.2 Hs.73722 APEX1 APEX nuclease5′-AGCTGAACTTCAGGAGCTGCC-3′ 194 (multifunctional DNA repair enzyme) 1NM_001641.2 Hs.73722 APEX1 APEX nuclease 5′-AAGCCTTTCGCAAGTTCCTGA-3′ 195(multifunctional DNA repair enzyme) 1 NM_001641.2 Hs.73722 APEX1APEX nuclease 5′-ACGGCATAGGCGATGAGGAG-3′ 196 (multifunctional DNA repairenzyme) 1 NM_001641.2 Hs.73722 APEX1 APEX nuclease5′-AGGAAGGCCGGGTGATTGTG-3′ 197 (multifunctional DNA repair enzyme) 1NM_001641.2 Hs.73722 APEX1 APEX nuclease 5′-GTCTGGTACGACTGGAGTA-3′ 198(multifunctional DNA repair enzyme) 1 NM_001641.2 Hs.73722 APEX1APEX nuclease 5′-GACAGCTTTAGGCACCTCTA-3′ 199 (multifunctional DNA repairenzyme) 1 NM_015641.2 Hs.533391 TES Testis derived transcript (35′-GGAUUCGAACUGCACUUCU-3′ 200 LIM domains) NM_015641.2 Hs.533391 TESTestis derived transcript (3 5′-ACUGUGGCACCCAGCUUGU-3′ 201 LIM domains)NM_003461.3 Hs.490415 ZYX Zyxin 5′-GCCCAAAGUGAAUCCCUUC-3′ 202NM_002880.2 Hs.159130 RAF1 V-raf-1 murine leukemia viral5′-TTTGAATATCTGTGCTGAGAACACA 203 oncogene homolog 1GTTCTCAGCACAGATATTCTTTTT-3′ NM_002880.2 Hs.159130 RAF1V-raf-1 murine leukemia viral 5′-TTTGTCAATTAGCTGGAACATCACAG 204oncogene homolog 1 ATGTTCCAGCTAATTGACTTTTT-3′ NM_004506.2 Hs.158195 HSF2Heat shock transcription 5′-AATGAGAAAAGCAAAAGGTGCCCTGT 205 factor 2CTC-3′ NM_005356.2 Hs.470627 LCK Lymphocyte-specific protein5′-CAUCGAUGUGUGUGAGAACUGC-3′ 206 tyrosine kinase NM_005546.3 Hs.483938ITK IL2-inducible T-cell kinase 5′-CUGUUCUCAGCUGGAGAAGCUU-3′ 207NM_005546.3 Hs.483938 ITK IL2-inducible T-cell kinase5′-GGAGCCUUCAUGGUAAGGGAUU-3′ 208 NM_002133.1 Hs.517581 HMOX1Heme oxygenase (decycling) 1 5′-GGCACCATGAAGGCG-3′ 209 NM_000639.1Hs.2007 FASLG Tumor necrosis factor (ligand)5′-CUGGGCU GUACU UUGUA UATT-3′ 210 superfamily, member 6 NM_018417.2Hs.320892 SAC Testicular soluble adenylyl 5′-AUGUAGCCUGGAGAUCCAUUU-3′211 cyclase NM_003743.3 Hs.412293 NCOA1 Nuclear receptor coactivator 15′-CCUCAGGGCAGAGAACCAUCUdTdT-3′ 212 NM_005572.2 Hs.491359 LMNA Lamin A/C5′-CUGGACUUCCAGAAGAACAUCdTdT-3′ 213 NM_176871.2 Hs.521444 PDLIM2PDZ and LIM domain 2 5′-AAGAUCCGCCAGAGCCCCUCG-3′ 214 (mystique)NM_014188.2 Hs.30026 HSPC182 HSPC182 protein 5′-AACAGGGACTCACGTGAAGCT-3′215 NM_014188.2 Hs.30026 HSPC182 HSPC182 protein5′-AAGACCTGTTTGATCTGATCC-3′ 216 AF263744.1 Hs.519346 ERBB2IPErbb2 interacting protein 5′-UAGACUGACCCAGCUGGAAdTdT-3′ 217 NM_002583.2Hs.406074 PAWR PRKC, apoptosis, WT1, 5′-GAUGCAAUUACACAACAGAdTdT-3′ 218regulator NM_003766.2 Hs.12272 BECN1 Beclin 1 (coiled-coil, myosin-5′-CUCAGGAGAGGAGCCAUUU-3′ 219 like BCL2 interacting protein) NM_003766.2Hs.12272 BECN1 Beclin 1 (coiled-coil, myosin- 5′-GAUUGAAGACACAGGAGGC-3′220 like BCL2 interacting protein) NM_004849.1 Hs.486063 APG5LAPG5 autophagy 5-like (S. 5′-GCAACUCUGGAUGGGAUUG-3′ 221 cerevisiae)NM_031482.3 Hs.527193 APG10L APG10 autophagy 10-like (S.5′-GGAGUUCAUGAGUGCUAUA-3′ 222 cerevisiae) NM_004707.2 Hs.264482 APG12LAPG12 autophagy 12-like (S. 5′-CAGAGGAACCUGCUGGCGA-3′ 223 cerevisiae)NM_002613.2 Hs.459691 PDPK1 3-phosphoinositide dependent5′-AACTGGCAACCTCCAGAGAAT-3′ 224 protein kinase-1 NM_002613.2 Hs.459691PDPK1 3-phosphoinositide dependent 5′-AAGAGACCTCGTGGAGAAACT-3′ 225protein kinase-1 NM_000314.2 Hs.500466 PTEN Phosphatase and tensin5′-AACAGTAGAGGAGCCGTCAAA-3′ 226 homolog (mutated in multipleadvanced cancers 1) NM_006092.1 Hs.405153 CARD4Caspase recruitment domain 5′-GGGUGAGACCAUCUUCAUCTT-3′ 227family, member 4 NM_006092.1 Hs.405153 CARD4 Caspase recruitment domain5′-GGCCAAAGUCUAUGAAGAUTT-3′ 228 family, member 4 NM_000598.3 Hs.450230IGFBP3 Insulin-like growth factor 5′-AAUCAUCAUCAAGAAAGGGCA-3′ 229binding protein 3 NM_006839.1 Hs.148559 IMMT Inner membrane protein,5′-AAUUGCUGGAGCUGGCCUUTT-3′ 230 mitochondrial (mitofilin) NM_016485.3Hs.431367 C6ORF55 Chromosome 6 open reading 5′-GAATGAAGATCGATAGTAA-3′231 frame 55 NM_016485.3 Hs.431367 C6ORF55 Chromosome 6 open reading5′-GCACAGGTGTAGCAAGTAA-3′ 232 frame 55 NM_016485.3 Hs.431367 C6ORF55Chromosome 6 open reading 5′-GGAGAATTATGCTTTGAAA-3′ 233 frame 55NM_016485.3 Hs.431367 C6ORF55 Chromosome 6 open reading5′-GCAGTGCTTTGCAGTATGA-3′ 234 frame 55 NM_016410.2 Hs.415534 SNF7DC2SNF7 domain containing 2 5′-CAGAAAGCCTTGCGAGTTT-3′ 235 NM_016410.2Hs.415534 SNF7DC2 SNF7 domain containing 2 5′-GAATTTGGATTGCCACA3A-3′ 236NM_016410.2 Hs.415534 SNF7DC2 SNF7 domain containing 25′-GAAGGTGTTCCCACTGATA-3′ 237 NM_016410.2 Hs.415534 SNF7DC2SNF7 domain containing 2 5′-GAGAGGGTCCTGCAAAGAA-3′ 238 NM_199185.1Hs.519452 NPM1 Nucleophosmin (nucleolar 5′-UGAUGAAAAUGAGCACCAG-3′ 239phosphoprotein B23, numatrin NM_003118.2 Hs.111779 SPARCSecreted protein, acidic 5-AAAATCCCTGCCAGAACCACC-3′ 240 cysteine-richNM_003118.2 Hs.111779 SPARC Secreted protein, acidic,5-AACAAGACCTTCGACTCTTCC-3′ 241 cysteine-rich NM_003183.3 Hs.404914ADAM17 A disintegrin and 5′-AAACGAAAGCGAGTACACT-3′ 242metalloproteinase domain 17 (tumor necrosis factor, alpha,converting enzyme) NM_012164.2 Hs.494985 FBXW2 F-box and WD-40 domain5′-AGATGGACTTCTCTGTACAGG-3′ 243 protein 2 NM_012164.2 Hs.494985 FBXW2F-box and WD-40 domain 5′-GACATTGTCTGTCTCTGAGGA-3′ 244 protein 2NM_175940.1 Hs.272813 DUOX1 Dual oxidase 1 5′-GGACUUAUCCUGGCUAGAGtt-3′245 NM_004503.2 Hs.820 HOXC6 Homeo box C6 5′ CCGGAUCUACUCGACUCCCTT 3′246 NM_004503.2 Hs.820 HOXC6 Homeo box C6 5′ CCUAAUCACACACUCUGLATT 3′247 NM_004503.2 Hs.820 HOXC6 Homeo box C6 5′ ACUGCAGACAAAACACCUUTT 3′248 NM_004503.2 Hs.820 HOXC6 Homeo box C6 5′ UCCAACCUCUGGGUCCGUUTT 3′249 NM_004503.2 Hs.820 H0XC6 Homeo box C6 5′ ACUGUGACCGUUUCUGUGUTT 3′250 NM_004503.2 Hs.820 HOXC6 Homeo box C6 5′ CUCAGACUCUACAGAUUGCTT 3′251 NM_182965.1 Hs.68061 SPHK1 Sphingosine kinase 15′-GGG CAA GGC CUU GCA GCU C-3′ 252 NM_003329.2 Hs.435136 TXNThioredoxin 5′-AUGACUGUCAGGAUGUUGCdTT-3′ 253 NM_003329.2 Hs.435136 TXNThioredoxin 5′-GCAACAUCCUGACAGUCAUdCC-3′ 254 NM_203500.1 Hs.465870 KEAP1Kelch-like ECH-associated 5′-UGAACGGUGCUGUCAUGUAdTdT-3′ 255 protein 1NM_005239.4 Hs.517296 ETS2 V-ets erythroblastosis virus5′-GCAGAGGUUCGGCAUGAAUdTdT-3′ 256 E26 oncogene homolog 2 (avian)NM_002067.1 Hs.515056 GNA11 Guanine nucleotide binding5′-AAGATGTTCGTGGACCTG1AC-3′ 257 protein (G protein), alpha 11 (Gq class)NM_004827.1 Hs.480218 ABCG2 ATP-binding cassette, sub-5′-AAGATGATTGTTCGTCCCTGCTAT 258 family G (WHITE), member 2AGTGAGTCGTATTA-3′ NM_000610.3 Hs.502328 CD44 CD44 antigen (homing5′-GAACGAAUCCUGAAGACAUCU-3′ 259 function and Indian blood group system)NM_003489.1 Hs.155017 NRIP1 Nuclear receptor interacting5′-GAAGGAAGCUUUGCUAGCU-3′ 260 protein 1 NM_004995.2 Hs.2399 MMP14Matrix metalloproteinase 14 5′-AAGCCTGGCTACAGCAATATGCCTGT 261 CTC-3′NM_022045.2 Hs.546363 MTBP Mdm2, transformed 3T3 cell5′ GGCUCAUUUGCACUCAAUU 3′ 262 double minute 2, p53 bindingprotein (mouse) binding protein, 104 kDa NM_002392.2 Hs.369849 MDM2Mdm2, transformed 3T3 cell 5′ GCCACAAAUCUGAUAGUAU 3′ 263double minute 2, p53 binding  protein (mouse) NM_170707.1 Hs.491359 LMNALamin A/C 5′ CUGGACUUCCAGAAGAACA 3′ 264 NM_004759.3 Hs.519276 MAPKAPK2Mitogen-activated protein 5′-UGACCAUCACCGAGUUUAUdTdT-3′ 265kinase-activated protein kinase 2 NM_001948.2 Hs.527980 DUTDUTP pyrophosphatase 5′-GATTATAGGAAATGTTG-3′ 266 NM_016022.1 Hs.108408APH-1A Likely ortholog of C. elegans 5′-AAGAAGGCAGATGAGGGGTTA-3′ 267anterior pharynx defective 1A NM_031301.2 Hs.511703 PSFLAnterior pharynx defective 1B- 5′-AACAAAGATGGACCAACACAG-3′ 268 likeBC007496.1 Hs.36915 SMAD3 SMAD, mothers against DPP5′-GGACGAGGUCUGCGUGAAUdTdT-3′ 269 homolog 3 (Drosophila) NM_182763.1Hs.532826 MCL1 Myeloid cell leukemia 5′-AAGAAACGCGGUAAUCGGACU-3′ 270sequence 1 (BCL2-related) NM_001022.3 Hs.438429 RPS19Ribosomal protein S19 5′-GCACAAAGAGCTTGCTCCCttcaagaga 271GGGAGCAAGCTCTTTGTGC-3′ NM_001022.3 Hs.438429 RPS19 Ribosomal protein S195′-GTCCGGGAAGCTGAAAGTCttcaagaga 272 GACTTTCAGCTTCCCGGAC-3′ NM_001022.3Hs.438429 RPS19 Ribosomal protein S19 5′-GAGATCTGGACAGAATCGCttcaagaga273 GCGATTCTGTCCAGATCTC-3′ NM_001400.2 Hs.154210 EDG1Endothelial differentiation, 5′-GGAGAACAGCATTAAACTG-3′ 274sphingolipid G-protein-coupled receptor, 1 NM_001001938.1 Hs.546252C9orf47 Chromosome 9 open reading 5′-GGTCAACATTCTGATGTCT-3′ 275 frame 47NM_021972.2 Hs.68061 SPHK1 Sphingosine kinase 15′-GGGCAAGGCCTTGCAGCTC-3′ 276 NM_016068.1 Hs.423968 TTC11Tetratricopeptide repeat 5′-GTACAATGATGACATCCGTAA-3′ 277 domain 11NM_016068.1 Hs.423968 TTC11 Tetratricopeptide repeat5′-GTACGTCCGCGGGTTGCTGCA-3′ 278 domain 11 NM_153831.2 Hs.395482 PTK2PTK2 protein tyrosine kinase 2 5′-AAGCAUGUGGCCUGCUAUGGA-3′ 279NM_003749.2 Hs.442344 IRS2 Insulin receptor substrate 25′-GATCCCGCCTCAACAACAACAACAACT 280 TCAAGAGAGTTGTTGTTGTTGTTGAGGTTTTTTGGAAA-3′ NM_000691.3 Hs.531682 ALDH3A1 Aldehyde dehydrogenase 35′-AAG AAG AGC UUC GAG ACU 281 family, member A1 UUC-3′ NM_000689.3Hs.76392 ALDH1A1 Aldehyde dehydrogenase 1 5′-AAC TGG GAG AGT ACG GTT 282family, member A1 TCC-3′ NM_000604.2 Hs.549034 FGFR1Fibroblast growth factor 5′-AAGTCGGACGCAACAGAGAAA-3′ 283receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome)NM_006006.3 Hs.171299 ZBTB16 Zinc finger and BTB domain5′-GGCCAACCAGAUGCGGCUGUU-3′ 284 containing 16 NM_006006.3 Hs.171299ZBTB16 Zinc finger and BTB domain 5′-GAUGUUUGACAUCCUCUUCUU-3′ 285containing 16 NM_004348.1 Hs.122116 RUNX2 Runt-related transcription5′-GGCUGCAAGCAGUAUUUACUU-3′ 286 factor 2 NM_004348.1 Hs.122116 RUNX2Runt-related transcription 5′-GGACAGAGUCAGAUUACAGUU-3′ 287 factor 2NM_014382.2 Hs.546361 ATP2C1 ATPase, Ca++ transporting,5′-AGCCACTGTGGAAGAAGTATATT-3′ 288 type 2C, member 1 NM_002083.2 Hs.2704GPX2 Glutathione peroxidase 2 5′-CCCUCUGGUUGGUGAUUCAdTdT-3′ 289(gastrointestinal) NM_002083.2 Hs.2704 GPX2 Glutathione peroxidase 25′-GGAUGAUGGCACCUUCCUAdTdT-3′ 290 (gastrointestinal) NM_000942.4Hs.434937 PP1B Peptidylprolyl isomerase B 5′-AATTGGAGATGAAGATGTAGG-3′291 (cyclophilin B) NM_003153.3 Hs.524518 STAT6Signal transducer and activator 5′-CAGUUCCGCCACUUGCCAAdTdT-3′ 292of transcription 6,  interleukin-4 induced NM_003153.3 Hs.524518 STAT6Signal transducer and activator 5′-AGCCUGGUGGACAUUUAUUdTdT-3′ 293of transcription 6,  interleukin-4 induced NM_003153.3 Hs.524518 STAT6Signal transducer and activator 5′-GAUGUGUGAAACUCUGAACdTdT-3′ 294of transcription 6,   interleukin-4 induced NM_003153.3 Hs.524518 STAT6Signal transducer and activator 5′-CAGAUGGGUAAGGAUGGCAdTdT-3′ 295of transcription 6,  interleukin-4 induced NM_002945.2 Hs.461925 RPA1Replication protein A1, 70 kDa 5′-AAGCACUAUCAUUGCGAAUCC-3′ 296NM_003169.2 Hs.437056 SUPT5H Suppressor of Ty 5 homolog5′-AACTGGGCGAGTATTACATGA-3′ 297 NM_003318.3 Hs.169840 TTKTTK protein kinase 5′-TGAACAAAGTGAGAGACAT-3′ 298 NM_007194.3 Hs.291363CHEK2 CHK2 checkpoint homolog 5′-AATGTGTGAATGACAACTACT-3′ 299 (S. pombe)NM_002358.2 Hs.533185 MAD2L1 MAD2 mitotic arrest deficient-5′-AATACGGACTCACCTTGCTTG-3′ 300 like 1 (yeast) NM_001401.3 Hs.126667EDG2 Endothelial differentiation, 5′-r(CCGCCGCUUCCAUUUUUCCU)d 301lysophosphatidic acid G- (TT)-3′ protein-coupled receptor, 2 NM_001401.3Hs.126667 EDG2 Endothelial differentiation, 5′-r(AGGAAAAAUGGAAGCGGCGGG)302 lysophosphatidic acid G- d(TT)-3′) protein-coupled receptor, 2NM_004448.2 Hs.446352 ERBB2 V-erb-b2 erythroblastic5′-CCUGGAACUCACCUACCUGdTdT-3′ 303 leukemia viral oncogene homolog 2NM_004448.2 Hs.446352 ERBB2 V-erb-b2 erythroblastic5′-CUACCUUUCUACGGACGUGdTdT-3′ 304 leukemia viral oncogene homolog 2NM_004448.2 Hs.446352 ERBB2 V-crb-b2 crythroblastic5′-GAUCCGGAAGUACACGAUGdTdT-3′ 305 leukemia viral oncogene homolog 2NM_014812.1 Hs.533635 KAB KARP-1-binding protein5′-GAAGGAAUCCUCCAAGUCA-3′ 306 NM_002737.2 Hs.531704 PRKCAProtein kinase C, alpha 5′-AAGCTCCATGTCACAGTACGA-3′ 307 NM_212535.1Hs.460355 PRKCB1 Protein kinase C, beta 1 5′-AAGCGCTGCGTCATGAATGTT-3′308 NM_138578.1 Hs.516966 BCL2L1 BCL2-like 1 5′-CTG CCT AAG GCG GAT TTG309 AAT-3′ NM_138578.1 Hs.516966 BCL2L1 BCL2-like 15′-GGC AGG CGA CGA GTT TGA 310 ACT-3′ NM_138578.1 Hs.516966 BCL2L1BCL2-like 1 5′-GTG CGT GGA AAG CGT AGA 311 CAA-3′ NM_004050.2 Hs.410026BCL2L2 BCL2-like 2 5′-GGC GGA GTT CAC AGC TCT 312 ATA-3′ NM_004050.2Hs.410026 BCL2L2 BCL2-like 2 5′-TG GGC ATA AGT GCT GAT  313 CTA-3′NM_004050.2 Hs.410026 BCl2L2 BCL2-like 2 5′-CTC GGT CCT GCG ATT ATT 314AAT-3′ NM_003443.1 Hs.433764 ZBTB17 Zinc finger and BTB domain5′-AAGGCCGAGATCAGCAAAGTTCAAGAG 315 containing 17ACTTTGCTGATCTCGGCCTTTTTTTT-3′ NM_003345.3 Hs.302903 UBE2IUbiquitin-conjugating enzyme 5′-GGC CAG CCA UCA CAA UCA  316E21 (UBC9 homolog, yeast) ATT-3′ NM_003345.3 Hs.302903 UBE2IUbiquitin-conjugating enzyme 5′-GGA ACU UCU AAA UGA ACC  317E2I (UBC9 homolog, yeast) ATT-3′ NM_016166.1 Hs.162458 PIAS1Protein inhibitor of activated 5′-GGU CCA GUU AAG GUU UUG  318 STAT, 1UTT-3′ NM_016166.1 Hs.62458 PIAS1 Protein inhibitor of activated5′-GGU UAC CUU CCA CCU ACA  319 STAT, 1 ATT-3′ NM_004068.2 Hs.518460AP2M1 Adaptor-related protein 5′-AAGUGGAUGCCUUUCGGGUCA-3′ 320complex 2, mu 1 subunit NM_004068.2 Hs.518460 AP2M1Adaptor-related protein 5′-AAGGAGAACAGUUCUUGCGGC-3′ 321complex 2, mu 1 subunit NM_004068.2 Hs.518460 AP2M1Adaptor-related protein 5′-AAGGUCCAGU-CAUUCCAAAUG-3′ 322complex 2, mu 1 subunit NM_001278.2 Hs.198998 CHUKConserved helix-loop-helix 5′-AGGAAGGACCUGUUGACCUUTT-3′ 323ubiquitous kinase NM_001556.1 Hs.413513 IKBKB Inhibitor of kappa light5′-UGGUGAGCUUAAUGAAUGATT-3′ 324 polypeptide gene enhancer inB-cells, kinase beta NM_021975.2 Hs.502875 RELAV-rel reticuloendotheliosis  5′-AGAGGACAUUGAGGUGUAUTT-3′ 325viral oncogene homolog A NM_000963.1 Hs.196384 PTGS2Prostaglandin-endoperoxide 5′-AACTGCTCAACACCGGAATTTTT-3′ 326 synthase 2NM_005427.1 Hs.192132 TP73 Tumor protein p735′-CCAUCCUGUACAACUUCAUGUG-3′ 327 NM_005157.2 Hs.431048 ABL1V-abl Abelson murine 5′-CAAUAAGGAAGAAGCCCUUTT-3′ 328leukemia viral oncogene homolog 1 NM_005157.2 Hs.431048 ABL1V-abl Abelson murine 5′-TTAUUCCUUCUUCGGGAAGUC-3′ 329leukemia viral oncogene homolog 1 NM_001168.1 Hs.514527 BIRC5Baculoviral IAP repeat- 5′-GGCUGGCUUCAUCCACUGCTT-3′ 330containing 5 (survivin) NM_002940.1 Hs.12013 ABCE1ATP-binding cassette, sub- 5′-CGAAGATGTTGACCTGGTC-3′ 331family E (OABP), member 1 NM_002940.1 Hs.12013 ABCE1ATP-binding cassette, sub- 5′-AGAGTTGTCCTGTAGTTCG-3′ 332family E (OABP), member 1 NM_004208.2 Hs.424932 PDCD8Programmed cell death 8 5′-GGAAAUAUGGGAAAGAUCCdTdT 333(apoptosis-inducing factor) NM_000115.1 Hs.82002 EDNRBEndothelin receptor type B 5′-GGAGACUUUCAAAUACAUCTTtt-3′ 334 NM_001712.2Hs.512682 CEACAM1 Carcinoembryonic antigen- 5′-AACCTTCTGGAACCCGCCCAC-3′335 related cell adhesion molecule 1 NM_001712.2 Hs.512682 CEACAM1Carcinoembryonic antigen- 5′-AATGTTGCAGAGGGGAAGGAG-3′ 336related cell adhesion molecule 1 NM_033284.1 Hs.436900 TBL1YTransducin (beta)-like 1Y- 5′-AAGAGAATGGAGCACATGAAA-3′ 337 linkedNM_033284.1 Hs.436900 TBL1Y Transducin (beta)-like 1Y-5′-AAGATGAGCATAACCAGTGAC-3′ 338 linked NM_024665.3 Hs.438970 TBL1XR1Transducin (beta)-like 1X- 5′-AAGGCCCTATATTTGCATTAA-3′ 339linked receptor 1 NM_173174.1 Hs.491322 PTK2BPTK2B protein tyrosine kinase 5′-GTTGGCTGAGTGCTATGGGCTGA-3′ 340 2 betaNM_006311.2 Hs.462323 NCOR1 Nuclear receptor5′-GGGCTTAT0GAGGACCCTATGA-3′ 341 co-repressor 1 NM_002211.2 Hs.429052ITGB1 Integrin, beta 1 5′-GGAACAGCAGAGAAGCTCATTCAAGAG 342ATGAGCTTCTCTGCTGTTCCTTTTT-3′ NM_139176.2 Hs.351118 NALP7NACHT, leucine rich repeat 5′-CACCGAAGCAGCACGACTTCTTCTTCA 343and PYD containing 7 AGAGAGAAGAAGTCGTGCTGCTTC-3′ NM_004422.2 Hs.118640DVL2 Dishevelled, dsh homolog 2 5′-AGGUUCAGCAGCUCCACGGAdTdT-3′ 344(Drosophila) NM_001228.2 Hs.369736 CASP8 Caspase 8, apoptosis-related5′-gatccccCCTCGGGGATACTGTCTGAt 345 cysteine proteasetcaagagaTCAGACAGTATCCCCGAGGttt ttggaaa-3′ NM_001769.2 Hs.114286 CD9CD9 antigen (p24) 5′-GAGCATCTTCGAGCAAGAA-3′ 346 NM_004357.3 Hs.512857CD151 CD151 antigen 5′-CATGTGGCACCGTTTGCCT-3′ 347 NM_003188.2 Hs.485968MAP3K7 Mitogen-activated protein 5′-UGGCUUAUCUUACACUGGA-3′ 348kinase kinase kinase 7 NM_006116.2 Hs.507681 MAP3K7IP1Mitogen-activated protein 5′-GGCUCAAGUUCAGGAGUGAGAACAA-3′ 349kinase kinase kinase 7 interacting protein 1 NM_015093.2 Hs.269775MAP3K7IP2 Mitogen-activated protein 5′-GGAACGACUUCAAAGAGAACUUGAG-3′ 350kinase kinase kinase 7 interacting protein 2 NM_001315.1 Hs.485233MAPK14 Mitogen-activated protein 5′-GCAUUACAACCAGACAGUUGAUAUU-3′ 351kinase 14 NM_006502.1 Hs.439153 POLH Polymerase (DNA directed),5′-GUG GAG CAG CGG CAA AAU  352 eta CTT-3′ NM_006502.1 Hs.439153 POLHPolymerase (DNA directed), 5′-UCC UCA UUU GAG GAA UAA  353 eta ATT-3′NM_006502.1 Hs.439153 POLH Polymerase (DNA directed),5′-GGA AUA AAC CUU GUG CAG  354 eta UTT-3′ NM_006502.1 Hs.439153 POLHPolymerase (DNA directed), 5′-UAA ACC UUG UGC AGU UGU  355 eta ATT-3′NM_006502.1 Hs.439153 POLH Polymerase (DNA directed),5′-CCU UGU GCA GUU GUA CAG  356 eta UTT-3′ NM_015321.1 Hs.371096 MECT1Mucoepidermoid carcinoma 5′-CCGGCAACCUCGCGGCCAAUU-3′ 357 translocated 1NM_181715.1 Hs.406392 TORC2 Transducer of regulated cAMP5′-CGACUACCAUCUGCACUUAUU-3′ 358 response element-bindingprotein (CREB) 2 NM_001079.3 Hs.234569 ZAP70 Zeta-chain (TCR) associated5′-AACCGGCTCTCCATTGGCATT-3′ 359 protein kinase 70 kDa NM_004834.3Hs.431550 MAP4K4 Mitogen-activated protein 5′-GTGGTTGGAAATGGCACCTTT-3′360 kinase kinase kinase kinase 4 NM_006191.1 Hs.524498 PA2G4Proliferation-associated 2G4, 5′-AAGCGACCAGGAUUAUAUUCU-3′ 361 38 kDaNM_006191.1 Hs.524498 PA2G4 Proliferation-associated 2G4,5′-AAGUGAGGUGGAAAGGCGUUU-3′ 362 38 kDa NM_005940.3 Hs.143751 MMP11Matrix metalloproteinase 11 5′-TCCCATGTCCACTTCGACTATGATGTCA 363(stromelysin 3) AGAGCATCATAGTCGAAGTGGACATTT-3′ NM_005940.3 Hs.143751MMP11 Matrix metalloproteinase 11 5′-TCCCAGATCTACTTCTTCCGAGGTCAAG 364(stromelysin 3) AGCCTCGGAAGAAGTAGATCTTT-3′ NM_005940.3 Hs.143751 MMP11Matrix metalloproteinase 11 5′-TCCCAGGATGCTGATGGCTATGCCTTCA 365(stromelysin 3) AGAGAGGCATAGCCATCAGCATCCTTT-3′ NM_003684.3 Hs.371594MKNK1 MAP kinase interacting 5′-AATGCCCATCTCTATAGGTTT-3′ 366serine/threonine kinase 1 NM_003668.2 Hs.413901 MAPKAPK5Mitogen-activated protein 5′-GGAUAUGCGAAGAAAGAUCTT-3′ 367kinase-activated protein kinase 5 NM_004604.3 Hs.83734 STX4ASyntaxin 4A (placental) 5′-AAGGAGGAAGCTGATGAGAAC-3′ 368 NM_004177.3Hs.530733 STX3A Syntaxin 3A 5′-AACGTCCGGAACAAACTGAAG-3′ 369NM_001009567.1 Hs.461247 MRC1L1 Mannose receptor, C type 1-5′-AAGTGGTACGCAGATTGCACG-3′ 370 like 1 NM_002576.3 Hs.435714 PAK1P21/Cdc42/Rac1-activated 5′-AAGGAGAAGAAAAAGAAGGAC-3′ 371 kinase 1NM_001664.2 Hs.247077 RHOA Ras homolog gene family,5′-GCAGGTAGAGTTGGCTTTG-3′ 372 member A NM_175744.3 Hs.502659 RHOCRas homolog gene family, 5′-GACTATGATCGACTGCGGC-3′ 373 member CNM_080491.1 Hs.429434 GAB2 GRB2-associated binding5′-GTGAGAACGATGAGAAATA-3′ 374 protein 2 NM_080491.1 Hs.429434 GAB2GRB2-associated binding 5′-GTTGGTGCCTAATCACTTA-3′ 375 protein 2NM_005225.1 Hs.96055 E2F1 E2F transcription factor 15′-GACGTGTCAGGACCTTCGT-3′ 376 NM_005225.1 Hs.96055 E2F1E2F transcription factor 1 5′-CTTAACTGGTGTACATTAA-3′ 377 NM_006392.2Hs.376064 NOL5A Nucleolar protein 5A (56 kDa5′-CAAUAUGAUCAUCCAGUCCAUUA-3′ 378 with KKE/D repeat) NM_015934 Hs.471104NOP5/NOP58 Nucleolar protein 5′-CAAGCAUGCAGCUUCUACCGUUC-3′ 379NOP5/NOP58 NM_001436 Hs.299002 FBL Fibrillarin5′-CAGUCGAGUUCUCCCACCGCUCU-3′ 380 NM_006666 Hs.515846 RUVBL2RuvB-like 2 (E. coli) 5′-GAGACCAUCUACGACCUGGGCAC-3′ 381 NM_006666Hs.515846 RUVBL2 RuvB-like 2 (E. coli) 5′-GAGAGUGACAUGGCGCCUGUCCU-3′ 382NM_003707.1 Hs.272822 RUVBL2 RuvB-like 1 (E. coli)5′-AAGGAACCAAACAGUUGAAACUG-3′ 383 NM_003707.1 Hs.272822 RUVBL2RuvB-like 1 (E. coli) 5′-GAGUCUUCUAUCGCUCCCAUCGU-3′ 384 NM_004741Hs.523238 NOLC1 Nucleolar and coiled-body 5′-AAAUUGAGGUGGAUUCACGAGUU-3′385 phosphoprotein 1 NM_032177 Hs.546453 PHAX RNA U, small nuclear RNA5′-UAGUAUCAGCGAGGAACAAAUUA-3′ 386 export adaptor(phosphorylation regulated) NM_032177 Hs.546453 PHAXRNA U, small nuclear RNA 5′-AAGAGUAUAUAGCACAGGAUUUA-3′ 387export adaptor (phosphorylation regulated) NM_024831 Hs.335068 NCOA6IPNuclear receptor coactivator 6 5′-AAGAUUGCCCUUGCUCGCAAUAA-3′ 388interacting protein NM_024831 Hs.335068 NCOA6IPNuclear receptor coactivator 6 5′-UAUCACCGUAUGAAAUGGAAACU-3′ 389interacting protein NM_022874.1 Hs.202179 SMN2Survival of motor neuron 1, 5′-AAGUGGAAUGGGUAACUCUUCUU-3′ 390 telomericNM_012321.2 Hs.515255 LSM4 LSM4 homolog, U6 small5′-AACGGCCGUCCCAAAGCUGGCUG-3′ 391 nuclear RNA associated NM_016200.2Hs.446179 LSM8 LSM8 homolog, U6 small 5′-AAGAAACAGAUUCUGCGCUUGAU-3′ 392nuclear RNA associated NM_003142 Hs.546301 SSBSjogren syndrome antigen B 5′-GAAUUAGGUCCACUUCAAUGUCC-3′ 393(autoantigen La) NM_003142 Hs.546301 SSB Sjogren syndrome antigen B5′-AAGAUUCUUCCAUUAAAUUGCCU-3′ 394 (autoantigen La) NM_001228 Hs.369736CASP8 Caspase 8, apoptosis-related 5′-AACTACCAGAAAGGTATACCT-3′ 395cysteine protease NM_003842.3 Hs.521456 TNFRSF10BTumor necrosis factor receptor 5′-AAGACCCTTGTGCTCGTTGTC-3′ 396superfamily, member 10b NM_017672.2 Hs.512894 TRPM7Transient receptor potential 5′-AAGCAGAGTGACCTGGTAGAT-3′ 397cation channel, subfamily M, member 7 NM_007294.1 Hs.194143 BRCA1Breast cancer 1, early onset 5′-UCACAGUGUCCUUUAUGUAdTdT-3′ 398NM_033238.1 Hs.526464 PML Promyelocytic leukemia5′-AUGGCUUCGACGAGUUCAATT-3′ 399 NM_000546.2 Hs.408312 TP53Tumor protein p53 (Li- 5′-GCAUGAACCGGAGGCCCAUTT-3′ 400Fraumeni syndrome) NM_002198.1 Hs.436061 IRF1Interferon regulatory factor 1 5′-AGACCAGAGCAGGAACAAGTT-3′ 401NM_024790.3 Hs.370147 FLJ22490 Hypothetical protein FLJ224905′-GAAGATTTGCGCAGTGGAC-3′ 402 NM_000546.2 Hs.408312 TP53Tumor protein P53 (Li- 5′-UGGUUCACUGAAGACCCAGUU-3′ 403Fraumeni syndrome) NM_002880.2 Hs.159130 RAF1V-raf-1 murine leukemia viral 5′-AUUCCUGCUCAAUGGAUUUdTdT-3′ 404oncogene homolog 1 NM_198400.1 Hs.1565 NEDD4 Neural precursor cell5′-TAGAGCCTGGCTGGGTTGTTTTG-3′ 405 expressed, developmentallydown-regulated 4 NM_015277.2 Hs.185677 NEDD4L Neural precursor cell5′-AACCACAACACAAAGTCACAG-3′ 406 expressed, developmentallydown-regulated 4-like NM_016931.2 Hs.371036 NOX4 NADPH oxidase 45′-AAACCGGCAGGAGUUUACCCAG-3′ 407 NM_005975.2 Hs.51133 PTK6PTK6 protein tyrosine kinase 6 5′-AAGGUGGCCAUUAAGGUGAUU-3′ 408NM_005531.1 Hs.380250 IFI16 Interferon, gamma-inducible5′-UCAGAAGACCACAAUCUAC-3′ 409 protein 16 NM_000633.1 Hs.150749 BCL2B-cell CLL/lymphoma 2 5′-GUGAAGUCAACAUGCCUGC-dTdT-3′ 410 NM_182981.1Hs.528383 OKL38 Pregnancy-induced growth 5′-CACCCUACACGAAGCCAGATT-3′ 411inhibitor NM_002961.2 Hs.81256 S100A4 S100 calcium binding protein5′-GGA CAG AUG AAG CUG CUU  412 A4 UTT-3′ NM_014585.3 Hs.529285 SLC40A1Solute carrier family 40  5′-GGTGGACAAGAATGCTAGAC-3′ 413(iron-regulated transporter), member 1 NM_014585.3 Hs.529285 SLC40A1Solute carrier family 40  5′-GAAGGATTGACCAGTTAACC-3′ 414(iron-regulated transporter), member 1 NM_014585.3 Hs.529285 SLC40A1Solute carrier family 40  5′-GCTTGAACATGAGCAAGAGC-3′ 415(iron-regulated transporter), member 1 NM_021127.1 Hs.96 PMAIP1Phorbol-12-myristate-13- 5′-AACTTCCGGCAGAAACTTCTG-3′ 416acetate-induced protein 1 NM_002467.2 Hs.202453 MYCV-myc myelocytomatosis viral 5′-GCCACAGCAUACAUCCUGUdTdT-3′ 417oncogene homolog (avian) NM_002187.2 Hs.674 IL12B Interleukin 12B5′-CGCACGCUAAUGCUGGCAU1TdT-3′ 418 NM_019887.3 Hs.169611 DIABLODiablo homolog (Drosophila) 5′-AAGCGGUGUUUCUCAGAATTGtt-3′ 419 NM_017563Hs.150725 IL17RD Interleukin 17 receptor D5′-GUCGG AGGGA AGACA GUGCT T-3′ 420 NM_017563 Hs.150725 IL17RDInterleukin 17 receptor D 5′-GCAUG UGAUU GCUGA CGCCT T-3′ 421NM_003142.2 Hs.546301 SSB Sjogren syndrome antigen B5Õ-AAGGCTTCCC AACTGATGC AA-3Õ 422 (autoantigen La) NM_003142.2 Hs.546301SSB Sjogren syndrome antigen B 5Õ-AAGCCAAG GAAGCAT TGGGTA-3Õ 423(autoantigen La) NM_003142.2 Hs.546301 SSB Sjogren syndrome antigen B5Õ-AAGTACTAGAA GGAGAGGTGG-3Õ 424 (autoantigen La) NM_006101 Hs.414407KNTC2 Kinctochore associated 2 5′-GTTCAAAAGCTGGATGATCTT-3′ 425 NM_145697Hs.234545 CDCA1 Cell division cycle associated 15′-AAGATACGGTCCAGAAGCTTA-3′ 426 NM_003550 Hs.209128 MAD1L1MAD1 mitotic arrest deficient- 5′-CCAGCGGCTCAAGGAGGTTTT-3′ 427 like 1NM_002358 Hs.533185 MAD2L1 MAD2 mitotic arrest deficient-5′-GAGTCGGGACCACAGTTTATT-3′ 428 like 1 NM_004336 Hs.469649 BUB1BUB1 budding uninhibited by 5′-TAGGCTAATTGTACTGCTCTT-3′ 429benzimidazoles 1 homolog NM_001211.4 Hs.36708 BUB1BBUB1 budding uninhibited by 5′-GGAGATCCTCTACAAAGGGTT-3′ 430benzimidazoles 1 homolog beta NM_016343.3 Hs.497741 CENPFCentromere protein F, 5′-AAGAGATGCTAATAGCAGTTT-3′ 431350/400 ka (mitosin) NM_001813 Hs.75573 CENPECentromere protein E, 312 kDa 5′-ACTCTTACTGCTCTCCAGTTT-3′ 432 NM_004217Hs.442658 AURKB Aurora kinase B 5′-CGAGACCTATCGCCGCATCGT-3′ 433NM_005030 Hs.329989 PLK1 Polo-like kinase 1 5′-GGGCGGCTTTGCCAAGTGCTT-3′434 NM_004104 Hs.83190 FASN Fatty acid synthase5′-CCCUGAGAUCCCAGCGCUGdTdT-3′ 435 NM_021975.2 Hs.502875 RELAV-rel reticuloendotheliosis 5′-GATCAATGGCTACACAGGA-3′ 436viral oncogene homolog A NM_033256 Hs.348037 PPP1R14AProtein phosphatase 1, 5′-ACCUGUCGAGGACUUCAUCdTdT-3′ 437regulatory (inhibitor) subunit 14A NM_177966.3 Hs.151293 2′-PDE2′-phosphodiesterase 5′-GUACAAGGUGGAGCGCAACdTdT-3′ 438 NM_015355Hs.462732 SUZ12 Suppressor of zeste 12 5′-CCCGGAAATTTCCCGTCCC-3′ 439homolog NM_015355 Hs.462732 SUZ12 Suppressor of zeste 125′-GAGATGACCTGCATTGCCC-3′ 440 homolog NM_016179.1 Hs.262960 TRPC4Transient receptor potential 5′-ACUCUUGGUUCAGAAAGGATT-3′ 441cation channel, subfamily C,  member 4 NM_000249 Hs.195364 MLH1MutL homolog 1, colon cancer, 5′-GGTTCACTACTAGTAAACT-3′ 442nonpolyposis type 2 NM_000534 Hs.111749 PMS1PMS1 postmeiotic segregation 5′-GGAATCTACTCGTTTGTAT-3′ 443 increased 1NM_002198 Hs.436061 IRF1 Interferon regulatory factor 15′-CCAAGAACCAGAGAAAAGATT-3′ 444 NM_002199.2 Hs.374097 IRF2Interferon regulatory factor 2 5′-CUCUUUAGAAACUGGGCAATT-3′ 445NM_000546.2 Hs.408312 TP53 Tumor protein p53 (Li-5′-AAGACTCCAGTGGTAATCTAC-3′ 446 Fraumeni syndrome) NM_000051 Hs.435561ATM Ataxia telangiectasia mutated 5′-TAGAGCTACAGAACGAAAG-3′ 447(includes complementation groups A, C and D) NM_000051 Hs.435561 ATMAtaxia telangiectasia mutated 5′-GAATGTGAACACCACCAAA-3′ 448(includes complementation groups A, C and D) NM_000051 Hs.435561 ATMAtaxia telangiectasia mutated 5′-CTACACAAATATTGAGGAT-3′ 449(includes complementation groups A, C and D) NM_000051 Hs.435561 ATMAtaxia telangiectasia mutated 5′-CTGTACTTCCATACTTGAT-3′ 450(includes complementation groups A, C and D) NM_001184 Hs.271791 ATRAtaxia telangiectasia and Rad3 5′-AAGCCAAGACAAATTCTGTGT-3′ 451 relatedNM_001184 Hs.271791 ATR Ataxia telangiectasia and Rad35′-AACCTCCGTGATGTTGCTTGA-3′ 452 related NM_001798.2 Hs.19192 CDK2Cyclin-dependent kinase 2 5′-CAAAGCCAGAAACAAGTTG-3′ 453 NM_001798.2Hs.19192 CDK2 Cyclin-dependent kinase 2 5′-AAATAAACTCTACCTGGTT-3′ 454NM_001798.2 Hs.19192 CDK2 Cyclin-dependent kinase 25′-AAACCTCAGAATCTGCTTA-3′ 455 NM_001798.2 Hs.19192 CDK2Cyclin-dependent kinase 2 5′-GTTACTTCTATGCCTGATT-3′ 456 NM_207003Hs.469658 BCL2L11 BCL2-like 11 (apoptosis 5′-(GACCGAGAAGGUAGACAAUUG) 457facilitator) d(TT)-3′ NM_000166 Hs.333303 GJB1Gap junction protein, beta 1, 5′-AAGAGGCACAAGGTCCACATCdTdT-3′ 458 32 kDaNM_000359 Hs.508950 TGM1 Transglutaminase 15′-AUGCAGCUGGAGAUGGCACdTdT-3′ 459 NM_024596 Hs.550532 MCPH1Microcephaly, primary 5′-AGGAAGUUGGAAGGAUCCAdTdT-3′ 460autosomal recessive 1 NM_024596 Hs.550532 MCPH1 Microcephaly, primary5′-GAACACUUAUCAAGCCUAAUU-3′ 461 autosomal recessive 1 NM_024596Hs.550532 MCPH1 Microcephaly, primary 5′-GGAGAGAACAAGCAUAUUUUU-3′ 462autosomal recessive 1 NM_024596 Hs.550532 MCPH1 Microcephaly, primary5′-UGAUGUACCUAUUCUCUUAUU-3′ 463 autosomal recessive 1 NM_024596Hs.550532 MCPH1 Microcephaly, primary 5′-GAUAAGAGAUUUCAGAAGAUU-3′ 464autosomal recessive 1 NM_024596 Hs.550532 MCPH1 Microcephaly, primary5′-GUCACCACAGCGCAATGGAdTdT-3′ 465 autosomal recessive 1 NM_000245Hs.132966 MET Met proto-oncogene 5′-ACUCUAGAUGCUCAGACUUTT-3′ 466(hepatocyte growth factor receptor) NM_205860.1 Hs.33446 NR5A2Nuclear receptor subfamily 5, 5′-AGGATCCATCTTCCTGGTTAC-3′ 467group A, member 2 NM_182763.1 Hs.532826 MCL1 Myeloid cell leukemia5′-UAACACCAGTACGGACGGGdTdT-3′ 468 sequence 1 (BCL2-related) NM_008765Hs.444870 ORC2L Origin recognition complex, 5′-UGCUCCUCUCAUGUGGGAU-3′469 subunit 2-like NM_006190 Hs.444870 ORC2L Origin recognition complex,5′-UCAUUGGUCAGUUGUCAUC-3′ 470 subunit 2-like NM_181837 Hs.410228 ORC3LOrigin recognition complex, 5′-GAGACUUGGGCGGUCAAAU-3′ 471 subunit 3-likeNM_002592.2 Hs.147433 PCNA Proliferating cell nuclear5′-CGGUGACACUCAGUAUGUC-3′ 472 antigen NM_016526 Hs.414418 BET1LBlocked early in transport 1 5′-AAGCAUGACCAGCCUGCUUAC-3′ 473homolog (S. cerevisiae) like NM_001569 Hs.522819 IRAK1Interleukin-1 receptor- 5′-GGUUGUCCUUGAGUAAUAAtt-3′ 474associated kinase 1 NM_080649 Hs.73722 APEX1 APEX nuclease5′-GUCUGGUACGACUGGAGUACC-3′ 475 (multifunctional DNA repair enzyme) 1NM_002658 Hs.77274 PLAU Plasminogen activator,5′-AACATTCACTGGTGCAACTGC-3′ 476 urokinase NM_001654 Hs.446641 ARAFV-raf murine sarcoma 3611 5′-AACAACATCTTCCTACATGAG-3′ 477viral oncogene homolog NM_004333 Hs.490366 BRAFV-raf murine sarcoma viral 5′-AAAGAATTGGATCTGGATCAT-3′ 478oncogene homolog B1 NM_002880 Hs.159130 RAF1V-raf-1 murine leukemia viral 5′-AAUAGUUCAGCAGUUUGGCUA-3′ 479oncogene homolog 1 NM_014314 Hs.190622 DDX58 DEAD (Asp-Glu-Ala-Asp) box5′-GAATTTAAAACCAGAATTATC-3′ 480 Polypeptide 58 NM_000927.3 Hs.489033ABCB1 ATP-binding cassette, sub- 5′-AAGCGAAGCAGTGGTTCAGGT-3′ 481family B (MDR/TAP), member 1 NM_001753.3 Hs.74034 CAV1Caveolin 1, caveolae protein, 5′-AGACGAGCUGAGCGAGAAGCA-3′ 482 22 kDaNM_001753.3 Hs.74034 CAV1 Caveolin 1, caveolae protein,5′-CAUCUACAAGCCCAACAACTT-3′ 483 22 kDa NM_000389.2 Hs.370771 CDKN1ACyclin-dependent kinase 5′-CUUCGACUUUGUCACCGAG-3′ 484inhibitor 1A (p21, Cip1) NM_007294.1 Hs.194143 BRCA1Breast cancer 1, early onset 5′-AACCTGTCTCCACAAAGTGTG-3′ 485 NM_002105Hs.477879 H2AFX H2A histone family, member X 5′-CAA CAA GAA GAC GCG AAU 486 CdTdT-3′ NM_020382 Hs.443735 SET8 PR/SET domain containing5′-AAUCGCCUAGGAAGACUGAUC-3′ 487 protein 8 NM_012331 Hs.490981 MSRAMethionine sulfoxidc reductase A 5′-CCCCUGUAGCGGCCAAACAUU-3′ 488NM_012331 Hs.490981 MSRA Methionine sulfoxide reductase A5′-CAAAGUACAAAGGAAUUUAUU-3′ 489 NM_012331 Hs.490981 MSRAMethionine sulfoxide reductase A 5′-CGGGAGGGACAGACUUUCUUU-3′ 490NM_014554 Hs.371957 SENP1 SUMO1/sentrin specific5′-GTGAACCACAACTCCGTATTC-3′ 491 protease 1 NM_002945 Hs.461925 RPA1Replication protein A1, 70 kDa 5′-AACUGGUUGACGAAAGUGGUG-3′ 492 NM_001184Hs.271791 ATR Ataxia telangiectasia and Rad3 5′-AACCCGCGUUGGCGUGGUUGA-3′493 related NM_001430.3 Hs.468410 EPAS1 Endothelial PAS domain5′-ACCAAUCCAGCACCCAUCCdTdT-3′ 494 protein 1 NM_001530.2 Hs.509554 HIF1AHypoxia-inducible factor 1, 5′-CUGAUGACCAGCAACUUGAdTdT-3′ 495alpha subunit (basic helix-loop- helix transcription factor) NM_021972Hs.68061 SPHK1 Sphingosine kinase 1 5′-GAGCUGCAAGGCCUUGCCCdTdT-3 496NM_002502 Hs.73090 NFKB2 Nuclear factor of kappa light5′-CTCCTCCATTGTGGAACCCAAGG 497 polypeptide gene enhancer in AGC-3′B-cells 2 (p49/p100) NM_016829 Hs.380271 OGG1 8-oxoguanine DNA5′-GUAUGGACACUGACUCAGAUU-3′ 498 glycosylase NM_016829 Hs.380271 OGG18-oxoguanine DNA 5′-GUACUUCCAGCUAGAUGUUUU-3′ 499 glycosylase NM_006142Hs.523718 SFN Stratifin 5′-GAGCGAAACCUGCUCUCAG-3′ 500 NM_006142Hs.523718 SFN Stratifin 5′-GGGUGACUACUACCGCUAC-3′ 501 NM_006142Hs.523718 SFN Stratifin 5′-AGACAGCACCCUCAUCAUG-3′ 502 NM_00615 Hs.477693NCK1 NCK adaptor protein 1 5′-GUCCUGGUGGC3AGUUCGATT-3′ 503 NM_00615Hs.477693 NCK1 NCK adaptor protein 1 5′-CGUCUCUAUGACCUCAACATT-3′ 504NM_002422 Hs.375129 MMP3 Matrix metalloproteinase 35′-AUGAAGAGUCUUCCAAUCCUU-3′ 505 (stromelysin 1, progelatinase)NM_000021.2 Hs.3260 PSEN1 Presenilin 1 (Alzheimer5′-AAGGTCCACTTCGTATGCTGG-3′ 506 disease 3) NM_015331 Hs.517249 NCSTNNicastrin 5′-AAGGGCAAGTTTCCCGTGCAG-3′ 507 NM_016022 Hs.108408 APH-1AAnterior pharynx defective 1 5′-AAGAAGGCAGATGAGGGGTTA-3′ 508homolog A (C. elegans) NM_172341 Hs.534465 PEN2Presenilin enhancer 2 homolog 5′-AAUCAAAGGCUAUGUCUGGCG-3′ 509(C. elegans) NM_020673 Hs.529044 RAB22A RAB22A, member RAS5′-AAGGACUACGCCGACUCUAUU-3′ 510 oncogene family NM_001002814 Hs.191179RAB11FIP1 RAB11 family interacting 5′-CGCCTCTTTCCCAGTCCATGT-3′ 511protein 1 (class I) NM_015470 Hs.24557 RAB11FIP5RAB11 family inetracting 5′-GAGCTGAGTGCTCAGGCTAAA-3′ 512protein 5 (class I) NM_030791 Hs.24678 SGPP1 Sphingosine-1-phosphate5′-AGUGGCCCGUUUCCAGCGGdTT-3′ 513 phosphatase 1 NM_005406 Hs.306307 ROCK1Rho-associated, coiled-coil 5′-AAGGTGATTGGTAGAGGTGCA-3′ 514containing protein kinase 1 NM_198437 Hs.250822 STK6Serine/threonine kinase 6 5′-AAGCACAAAAGCTTGTCTCCA-3′ 515 NM_006272Hs.422181 S100B S100 calcium binding protein, 5′-GGAAUUCAUGGCCUUUGUU-3′516 beta (neural) NM_004219 Hs.350966 PTTG1Pituitary tumor-transforming 1 5′-GAU CUC AAG UUU CAA CAC  517 Ctt-3′NM_004219 Hs.350966 PTTG1 Pituitary tumor-transforming 15′-GUC UGU AAA GAC CAA GG  518 GAtt-3′ NM_001478.2 Hs.159481 GALGTUDP-N-acetyl-alpha-D- 5′-GGAGCAAGUAGUGGGGCUGdTdT-3′ 519galactosamine:(N- acetylneuraminyl)- galactosylglueosylceramide N-acetylgalactosaminyltransferase NM_000657 Hs.150749 BCL2B-cell CLL/lymphoma 2 5′-GUACAUCCAUUAUAAGCUGTT-3′ 520 NM_032984Hs.368982 CASP2 Caspase 2, apoptosis-related5′-AACTTCCAGCTGGCATATAGGdTdT-3′ 521 cysteine protease NM_001228Hs.369736 CASP8 Caspase 8, apoptosis-related5′-AAGGGUCAUGCUCUAUCAGAUdTdT-3′ 522 cysteine protease NM_197967Hs.474150 BID BH3 interacting domain death5′-AAGAAGACAUCAUCCGGAAUAdTdT-3′ 523 agonist NM_001167 Hs.356076 BIRC4Baculoviral IAP repeat- 5′-AAGGAGAUACCGUGCGGUGCUdTdT-3′ 524 containing 4NM_002483 Hs.466814 CEACAM6 Carcinoembryonic antigen-5′-CCGGACAGTTCCATGTATA-3′ 525 related cell adhesion molecule 6NM_001008490 Hs.285313 KLF6 Kruppel-like factor 65′-GGAGAAAAGCCUUACAGAUTT-3′ 526 NM_024309 Hs.368551 TNIP2TNFAIP3 interacting protein 2 5′-GUAUUUGGCCGCCGACGCAd(TT)-3′ 527NM_001621 Hs.171189 AHR Aryl hydrocarbon receptor5′-AAGACTGGAGAAAGTGGCATG-3′ 528 NM_001005845 Hs.2442 ADAM9A disintegrin and 5′-AAUCACUGUGGAGACAUUUGCdTdT-3′ 529metalloproteinase domain 9 (meltrin gamma) NM_001110 Hs.172028 ADAM10A disintegrin and 5′-AAUGAAGAGGGACACUUCCCUdTdT-3′ 530metalloproteinase domain 10 NM_021641 Hs.386283 ADAM12 A disintegrin and5′-AACCUCGCUGCAAAGAAUGUGdTdT-3′ 531 metalloproteinase domain 12NM_207196 Hs.312098 ADAM15 A disintegrin and5′-AACUCCAUCUGUUCUCCUGACdTdT-3′ 532 metalloproteinase domain 15(metargidin) NM_021832 Hs.404914 ADAM17 A disintegrin and5′-AAAGUUUGCUUGGCACACCUUdTdT-3′ 533 metalloproteinase domain 17NM_000927.3 Hs.489033 ABCB1 ATP-binding cassette, sub-5′-AAG GCC TAA TGC CGA ACA  534 family B (MDR/TAP), member 1 CA-3′NM_000927.3 Hs.489033 ABCB1 ATP-binding cassette, sub-5′-AAC TTT GGC TGC CAT CAT  535 family B (MDR/TAP), member 1 CCA-3′NM_000572 Hs.193717 IL10 Interleukin 10 5′-UAAGCUCCAAGAGAAAGGCdTdT-3′536 NM_021975 Hs.502875 RELA V-rel reticuloendotheliosis 5′-GCCCUAUCCCUUUACGUCA-3′ 537 viral oncogene homolog A NM_001331Hs.166011 CTNND1 Catenin (cadherin-associated5′-GTGGACCATGCACTGCATGCCTAT 538 protein), data 1 AGTGAGTCGTATTAC-3′NM_001211 Hs.36708 BUB1B BUB1 budding uninhibited by5′- AGATCCTGGCTAACTGTTC-3′ 539 benzimidazoles 1 homolog beta NM_002358Hs.533185 MAD2L1 MAD2 mitotic arrest deficient-5′-TACGGACTCACCTTGCTTG-3′ 540 like 1 (yeast) NM_001530.2 Hs.509554 HIF1AHypoxia-inducible factor 1, 5′-CUGGACACAGUGUGUUUGAdTdT-3′ 541alpha subunit NM_001530.2 Hs.509554 HIF1A Hypoxia-inducible factor 1,5′-CUGAUGACCAGCAACUUGAdTdT-3′ 542 alpha subunit NM_001430 Hs.468410EPAS1 Endothelial PAS domain 5′-GCUCUUCGCCAUGGACACAdTdT-3′ 543 protein 1NM_001430 Hs.468410 EPAS1 Endothelial PAS domain5′-GCGACAGCUGGAGUAUGAAdTdT-3′ 544 protein 1 NM_001379 Hs.202672 DNMT1DNA (cytosine-5-)- 5′-CCAUGAGCACCGUUCUCCTT-3′ 545 methyltransferase 1NM_031310 Hs.107125 PLVAP Plasmalemma vesicle5′-CUUGACCAAGGAGCUCAACTT-3′ 546 associated protein NM_031310 Hs.107125PLVAP Plasmalemma vesicle 5′-GGAGCUCAACUUCACCACCTT-3′ 547associated protein NM_016734 Hs.126365 PAX5 Paired box gene 5 (B-cell5′-CGGCCACUCGCUUCCGGGCTT-3′ 548 lineage specific activator) NM_016734Hs.126365 PAX5 Paired box gene 5 (B-cell 5′-GCUCCGUCGACUGCGCGCC-3′ 549lineage specific activator) NM_006257 Hs.498570 PRKCQProtein kinase C, theta 5′-AAACCACCGTGGAGCTCTACT-3′ 550 NM_006257Hs.498570 PRKCQ Protein kinase C, theta 5′-AAGAGCCCGACCTTCTGTGAA-3′ 551NM_032430 Hs.182081 BRSK1 BR serine/threonine kinase 15′-GUU CUU CCG CCA GAU UGU  552 GdTdT-3′ NM_015045 Hs.203099 KIAA0261KIAA0261 5′-CGGACUACCCUUAGCACAAUU-3′ 553 NM_015045 Hs.203099 KIAA0261KIAA0261 5′-GAAUAGUCACCAUAUUCACUU-3′ 554 NM_005430 Hs.248164 WNT1Wingless-type MMTV 5′-GGTTCCATCGAATCCTGCA-3′ 555integration site family, member 1 NM_004421.2 Hs.74375 DVL1Dishevelled, dsh homolog 1 5′-AACAAGATCACCTTCTCCGAG-3′ 556 (Drosophila)NM_004422 Hs.118640 DVL2 Dishevelled, dsh homolog 25′-AACTTTGAGAACATGAGCAAC-3′ 557 (Drosophila) NM_139049 Hs.522924 MAPK8Mitogen-activated protein 5′-CGTGGATTTATGGTCTGTG-3′ 558 kinase 8NM_003376 Hs.73793 VEGF Vascular endothelial growth5′-UGGAUGUCUAUCAGCGCAGdTdT-3′ 559 factor NM_003376 Hs.73793 VEGFVascular endothelial growth 5′-GCUACUGCCAUCCAAUCGAdTdT-3′ 560 factorNM_003376 Hs.73793 VEGF Vascular endothelial growth5′-GGAGUACCCUGAUGAGAUCdTdT-3′ 561 factor NM_003376 Hs.73793 VEGFVascular endothelial growth 5′-CUGAGGAGUCCAACAUCACdTdT-3′ 562 factorNM_003376 Hs.73793 VEGF Vascular endothelial growth5′-CCAAGGCCAGCACAUAGGAdTdT-3′ 563 factor NM_005123 Hs.282735 NR1H4Nuclear receptor subfamily 1, 5′-GTCGTGACTTGCGACAAG-3′ 564group H, member 4 NM_004999 Hs.149387 MYO6 Myosin VI5′-GCUGGCAGUUCAUAGGAAUdTdT-3′ 565 NM_004999 Hs.149387 MYO6 Myosin VI5′-CGUGCUCCAAAGUCUGU1AdTdT-3′ 566 NM_014865 Hs.5719 CNAP1Chromosome condensation- 5′-UCAGUAUGUUGUGCAAGAGTT-3′ 567related SMC-associated protein 1 NM_014865 Hs.5719 CNAP1Chromosome condensation- 5′-GAAGAUACUCUGGAAUUCCTT-3′ 568related SMC-associated protein 1 NM_015261 Hs.438550 KIAA0056KIAA0056 protein 5′-CUGGAUUUCACAGAGACUGTT-3′ 569 NM_015261 Hs.438550KIAA0056 KIAA0056 protein 5′-GCAGAGAUCAUAGAGACUGTT-3′ 570 NM_015341Hs.308045 BRRN1 Barren homolog (Drosophila) 5′-GACUUUCCUCAGAAUGACGTT-3′571 NM_015341 Hs.308045 BRRN1 Barren homolog (Drosophila)5′-CAUUACUCCACCUGUAUCATT-3′ 572 NM_014551 Hs.180903 384D8-2hypothetical protein 384D8_6 5′-GGAUUUCAGGAUGAACACGTT-3′ 573 NM_014551Hs.180903 384D8-2 hypothetical protein 384D8_65′-GCUGCAGGACUUCCACCAGTT-3′ 574 NM_006031 Hs.474069 PCNT2Pericentrin 2 (kendrin) 5′-AAUUGGAACAGCUGCAGCAGA-3′ 575 NM_006031Hs.474069 PCNT2 Pericentrin 2 (kendrin) 5′-AAGCUCUGAUUUAUCAAAAGA-3′ 576NM_012179.2 Hs.5912 FBXO7 F-box protein 7 5′-CCCACACCAUUCCAUUCUA-3′ 577NM_002467 Hs.202453 MYC V-myc myelocytomatosis viral5′-AAGAUGAGGAAGAAAUCGAUGUU-3′ 578 oncogene homolog (avian) NM_002467Hs.202453 MYC V-myc myelocytomatosis viral 5′-AAAAGGUCAGAGUCUGGAUCACC-3′579 oncogene homolog (avian) NM_002467 Hs.202453 MYCV-myc myelocytomatosis viral 5′-CACGUCUCCACACAUCAGCACAA-3′ 580oncogene homolog (avian) NM_002467 Hs.202453 MYCV-myc myelocytomatosis viral 5′-AAAUGAGAUAAAGGUGGCUAAUU-3′ 581oncogene homolog (avian) NM_002392 Hs.369849 MDM2Mdm2, transformed 3T3 cell 5′-UGGUUGCAUUGUCCAUGGC-3′ 582double minute 2, p53 binding protein NM_003121 Hs.437905 SPIBSpi-B transcription factor 5′-GATCGCTGTGTGTCTGTAA-3′ 583(Spi-1/PU.1 related) NM_003120.1 Hs.502511 SPI1Spleen focus forming virus 5′-GTCCGTATGTAAATCAGAT-3′ 584(SFFV) proviral integration oncogene spi1 NM_199002 Hs.278186 ARHGEF1Rho guanine nucleotide 5′-CATACCATCTCTACCGACG-3′ 585exchange factor (GEF) 1 NM_014784 Hs.516954 ARHGEF11Rho guanine nucleotide 5′-ACTGAAGTCTCGGCCAGCT-3′ 586exchange factor (GEF) 11 NM_015313 Hs.24598 ARHGEF12Rho guanine nucleotide 5′-GAAACTCGTCGCATCTTCC-3′ 587exchange factor (GEF) 12 NM_173842 Hs.81134 IL1RN Interleukin 1 receptor5′-AUCUGCAGAGGCCUCCGCAtt-3′ 588 antagonist NM_032726 Hs.549218 PLCD4Phospholipase C, delta 4 5′-GAGCAGAACCTTCAGAATAdTdT-3′ 589 NM_032726Hs.549218 PLCD4 Phospholipase C, delta 4 5′-GAGCAGGGCTTCACCATTGdTdT-3′590 NM_032726 Hs.549218 PLCD4 Phospholipase C, delta 45′-GGAAGGAGAAGAATTCGTAdTdT-3′ 591 NM_032726 Hs.549218 PLCD4Phospholipase C, delta 4 5′-GATATCATCTTTCTCTGAAdTdT-3′ 592 NM_004104Hs.83190 FASN Fatty acid synthase 5′-CAACTACGGCTTTGCCAAT-3′ 593NM_004104 Hs.83190 FASN Fatty acid synthase 5′-GCAACTCACGCTCCGGAAA-3′594 NM_004104 Hs.83190 FASN Fatty acid synthase5′-GCCCTGAGCTGGACTACTT-3′ 595 NM_004104 Hs.83190 FASNFatty acid synthase 5′-GGTATGCGACGGGAAAGTA-3′ 596 NM_002165.2 Hs.504609ID1 Inhibitor of DNA binding 1, 5′-AACTCGGAATCCGAAGTTGGA-3′ 597dominant negative helix-loop- helix protein NM_003200.1 Hs.371282 TCF3Transcription factor 3 5′-AAAGACCTGAGGGACCGGGAG-3′ 598 NM_015895Hs.234896 GMNN Geminin, DNA replication 5′-GAGAAAATGAGCTGTCCGC-3′ 599inhibitor NM_015895 Hs.234896 GMNN Geminin, DNA replication5′-CTGGCAGAAGTAGCAGAAC-3′ 600 inhibitor NM_006704 Hs.281902 SUGT1SGT1, suppressor of G2 allele 5′-AAGGCUUUGGAACAGAAACCA-3′ 601of SKP1 (S. cerevisiae) NM_002358 Hs.533185 MAD2L1MAD2 mitotic arrest deficient- 5′-AAGAGUCGGGACCACAGUUUA-3′ 602 like 1NM_006472 Hs.533977 TXNIP Thioredoxin interacting5′-ACAGACUUCGGAGUACCUG1TT-3′ 603 protein NM_001379 Hs.202672 DNMT1DNA (cytosine-5-)- 5′-CGGUGCUCAUGCUUACAACTT-3′ 604 methyltransferase 1NM_001379 Hs.202672 DNMT1 DNA (cytosine-5-)- 5′-CGAGUUGCUAGACCGCUUCTT-3′605 methyltransferase 1 NM_006838 Hs.444986 METAP2Methionyl aminopeptidase 2 5′-AAUGCCGGUGACACAACAUGA-3′ 606 NM_007862.2Mm.382 Dlgh1 Discs, large homolog 1 5′-TACGGGAGCAGATGATGAAA-3′ 607(Drosophila) NM_007862.2 Mm.382 Dlgh1 Discs, large homolog 15′-AACCCAAATCCATGGAAAATA-3′ 608 (Drosophila) NM_008173.1 Mm.129481 Nr3c1Nuclear receptor subfamily 3, 5′-GAGCAGTGGAAGGACAGCATTCAA 609group C, member 1 GAGATGCTGTCCTTCCACTGCTCTTTT TT-3′ NM_008173.1Mm.129481 Nr3c1 Nuclear receptor subfamily 3,5′-GATCCCGAGCAGTGGAAGGACAGC 610 group C, member 1ATTCAAGAGATGCTGTCCTTCCACTGC TCTTTTTTGGAAA-3′ NM_007871.1 Mm.39292 Dnm2Dynamin 2 5′-GGACCAGGCAGAGAATGAG-3′ 611 NM_011155.1 Mm.3294 Ppp5cProtein phosphatase 5, 5′-AAG ACA CAG GCC AAC GAC 612 catalytic subunitUAC-3′ NM_011155.1 Mm.3294 Ppp5c Protein phosphatase 5,5′-AAG AUU GUG AAG CAG AAG 613 catalytic subunit GCC-3′ NM_009263.1Mm.288474 Spp1 Secreted phosphoprotein 1 5′-AATCTCCTTGCGCCACAGAAT-3′ 614NM_009263.1 Mm.288474 Sppl Secreted phosphoprotein 15′-AAGTCAGCTGGATGAACCAAG-3′ 615 NM_145978.1 Mm.283968 Pdlim2PDZ and LIM domain 2 5′-AAGAUCCGACAGAGCGC05CA-3′ 616 NM_146386.1Mm.32257 Myocd Myocardin 5′-AATGCAACTGCAGAAGCAGAA-3′ 617 NM_145541.3Mm.333868 Rap1a RAS-related protein-1a 5′-AAGCAAGTCGAGGTAGATTGC-3′ 618NM_010026.1 Mm.277236 Ddef1 RIKEN cDNA 1700010G065′-CAGCUAACUGCACUCCGAG-3′ 619 gene NM_010026.1 Mm.277236 Ddef1RIKEN cDNA 1700010G06 5′-UGAUAUUAUGGAAGCAAAU-3′ 620 gene NM_007614.2Mm.291928 Catnb Catenin beta 5′-AAGGCTTTTCCCAGTCCTTCA-3′ 621 NM_007614.2Mm.291928 Catnb Catenin beta 5′-AAGATGATGGTGTGCCAAGTG-3′ 622 NM_011952.1Mm.8385 Mapk3 Mitogen activated protein 5′-AATGTTATAGGCATCCGAGAC-3′ 623kinase 3 NM_011952.1 Mm.8385 Mapk3 Mitogen activated protein5′-AACCCAAACAAGCGCATCACA-3′ 624 kinase 3 NM_011949.2 Mm.196581 Mapk1Mitogen activated protein 5′-AAAGTTCGAGTTGCTATCAAG-3′ 625 kinase 1NM_010591.1 Mm.275071 Jun Jun oncogene 5′-GCGCATGAGGAACCGCATT-3′ 626NM_008416.1 Mm.1167 Junb Jun-B oncogene 5′-GACCAGGAGCGCATCAAAG-3′ 627NM_010592.3 Mm.1175 Jund1 Jun proto-oncogene related5′-AAGCCAGAACACCGAGCTG-3′ 628 gene d1 NM_010234.2 Mm.246513 FosFBJ osteosarcoma related 5′-GCGGAGACAGATCAACTTG-3′ 629 oncogeneNM_010235.1 Mm.6215 Fosl1 Fos-like antigen 1 5′-ATTGGAGGATGAGAAATCG-3′630 NM_008037.3 Mm.24684 Fosl2 Fos-like antigen 25′-TCAACGCCATCACCACCAG-3′ 631 NM_009505.2 Mm.282184 VegfaVascular endothelial growth 5′-AACGAUGAAGCCCUGGAGUGC-3′ 632 factor ANM_015774.2 Mm.264435 Ero11 ERO1-like (S. cerevisiae)5′-ACTTCATCAGAATGGCAGGGTTT-3′ 633 NM_021450.1 Mm.244705 Trpm7Transient receptor potential 5′-AACCGGAGGTCAGGTCGAAAT-3′ 634cation channel, subfamily M, member 7 NM_021450.1 Mm.244705 Trpm7Transient receptor potential 5′-AAGCAGAGTGACCTGGTAGAT-3 635cation channel, subfamily M, member 7 NM_177407.2 Mm.131530 Camk2aCalcium/calmodulin-dependent 5′-CACCACCAUUGAGGACGAAdTdT-3′ 636protein kinase II alpha NM_010118.1 Mm.290421 Egr2Early growth response 2 5′-GUGACCACCUUACUACUCAdTdT-3′ 637 NM_010118.1Mm.290421 Egr2 Early growth response 2 5′-GUUUGCCAGGAGUGACGAAdTdT-3′ 638NM_015806.2 Mm.18856 Mapk6 Mitogen-activated protein5′-GGCUUUUCAUGUAUCAGCUTT-3′ 639 kinase 6 NM_015806.2 Mm.18856 Mapk6Mitogen-activated protein 5′-GGCAAUGGCUUGGUUUUUUTT-3′ 640 kinase 6NM_015806.2 Mm.18856 Mapk6 Mitogen-activated protein5′-GGAGUACAUGGAGACAGACTT-3′ 641 kinase 6 NM_009744.2 Mm.347398 Bcl6B-cell leukemia/lymphoma 6 5′-GTCGAGACATCTTGACTGA-3′ 642 NM_009744.2Mm.347398 Bcl6 B-cell leukemia/lymphoma 6 5′-GACACGGATCTGAGAATCT-3′ 643NM_145533.1 Mm.136586 Smox Spermine oxidase 5′-GGACGUGGUUGAGGAAUUC-3′644 NM_008778.1 Mm.40035 Pak3 P21 (CDKN1A)-activated5′-TAGCAGCACATCAGTCGAATA-3′ 645 kinase 3 NM_008778.1 Mm.40035 Pak3P21 (CDKN1A)-activated 5′-CCCAATATTGTCAATTATTTA-3′ 646 kinase 3NM_080428.2 Mm.196475 Fbxw7 F-box and WD-40 domain5′-CACAAAGCTGGTGTGTGCA-3′ 647 protein 7, archipelago homolog NM_021450.1Mm.244705 Trpm7 Transient receptor potential 5′-AACCGGAGGTCAGGTCGAAAT-3′648 cation channel, subfamily M,  member 7 NM_021450.1 Mm.244705 Trpm7Transient receptor potential 5′-AAGCAGAGTGACCTGGTAGAT-3′ 649cation channel, subfamily M, member 7 NM_009505.2 Mm.282184 VegfaVascular endothelial growth 5′-AAGCCGTCCTGTGTGCCGCTG-3′ 650 factor ANM_009505.2 Mm.282184 Vegfa Vascular endothelial growth5′-AACGATGAAGCCCTGGAGTGC-3′ 651 factor A NM_010228.2 Mm.3464 Flt1FMS-like tyrosine kinase 1 5′-AAGTTAAAAGTGCCTGAACTG-3′ 652 NM_010228.2Mm.3464 Flt1 FMS-like tyrosine kinase 1 5′-AAGCAGGCCAGACTCTCTTTC-3′ 653NM_010612.2 Mm.285 Kdr Kinase insert domain protein5′-AAGCTCAGCACACAGAAAGAC-3′ 654 receptor NM_010612.2 Mm.285 KdrKinase insert domain protein 5′-AATGCGGCGGTGGTGACAGTA-3′ 655 receptorAK019429.1 Mm.3049 Cks1b CDC28 protein kinase 1b5′-GGGACATAGCCAAGCTGGTCgagt 656 actgGACCAGCTTGGCTATGTCC-3′ NM_008000.1Mm.280819 Fert2 Fer (fms/fps related) protein 5′-AAC TAC GGT TGC TGG AGA657 kinase, testis specific 2 CAG-3′ NM_009750.1 Mm.90787 Ngfrap1Nerve growth factor receptor 5′-CAACAACCACAACCATAAC-3′ 658(TNFRSF16) associated protein 1 NM_009750.1 Mm.90787 Ngfrap1Nerve growth factor receptor 5′-CATAACCACAACCACCACTdTdT-3′ 659(TNFRSF16) associated protein 1 NM_010431.1 Mm.3879 Hif1aHypoxia inducible factor 1, 5′-TGTGAGCTCACATCTTGAT-3′ 660 alpha subunitNM_010838 Mm.1287 Mapt Microtubule-associated protein5′-CCAGGAGTTTGACACAATG-3′ 661 tau NM_009045 Mm.2499661 RelaV-rel reticuloendotheliosis 5′-GATCAATGGCTACACAGGA-3′ 662viral oncogene homolog A NM_009689 Mm.8552 Birc5 Baculoviral IAP repeat-5′-GAGCCAAGAACAAAATTGC-3′ 663 containing 5 NM_009689 Mm.8552 Birc5Baculoviral IAP repeat- 5′-GAAAGTGCGCCGTGCCATC-3′ 664 containing 5NM_007798.1 Mm.236553 CTSB Cathepsin B 5′-CCACUGUGGCAUUGAAUCATT-3′ 665NM_011960 Mm.15962 Parg Poly (ADP-ribose) 5′-AACGCCACCTCGTTTGTTTTC-3′666 glycohydrolase NM_010928.1 Mm.254017 Notch2 Notch gene homolog 25′-GAUGUGGACAGUGUCUGUTT-3′ 667 NM_019984 Mm.41964 Tgm1Transglutaminase 1, K 5′-AUGCAGCUGGAGAUGGCACdTdT-3′ 668 polypeptideNM_009593 Mm.15691 Abcg1 ATP-binding cassette, sub-5′-CGTGGATGAGGTTGAGACA-3′ 669 family G (WHITE), member 1 NM_009593Mm.15691 Abcg1 ATP-binding cassette, sub- 5′-GGTGGACAACAACTTCACA-3′ 670family G (WHITE), member 1 NM_138955 Mm.101876 Abcg4ATP-binding cassette, sub- 5′-GAAGGTGGAGAACCATATC-3′ 671family G (WHITE), member 4 NM_138955 Mm.101876 Abcg4ATP-binding cassette, sub- 5′-GCACTTGAACTACTGGTAT-3′ 672family G (WHITE), member 4 NM_011658 Mm.3280 Twist1 Twist gene homolog 15′-AAGCTGAGCAAGATTCAGACC-3′ 673 NM_011658 Mm.3280 Twist1Twist gene homolog 1 5′-AGGTACATCGACTTCCTGTAC-3′ 674 NM_011658 Mm.3280Twist1 Twist gene homolog 1 5′-AGCGGGTCATGGCTAACGTGC-3′ 675 NM_009537Mm.3868 Yy1 YY1 transcription factor 5′-GGGAGCAGAAGCAGGUGCAGAU-3′ 676NM_172689 Mm.86382 Ddx58 DEAD (Asp-Glu-Ala-Asp) box5′-GCCCATTGAAACCAAGAAATT-3′ 677 polypeptide 58 NM_008500.1 Mm.12881 Lhx6LIM homeobox protein 6 5′-AGACGCAGAGGCCTTGGTTCAAGAGA 678CCAAGGCCTCTGCGTCTGACTTTTTC-3′ NM_009308 Mm.233846 Syt4 Synaptotagmin 45′-r(GAAGCACAGAGUGAAGACCA) 679 d(TT)-3′ NM_009308 Mm.233846 Syt4Synaptotagmin 4 5′-r(CGAGCAGGAGAACAGCGAG) 680 d(TT)-3′ NM_007615.1Mm.35738 Catns Expressed sequence AI225934 5′-GATGGTTATCCAGGTGGCA-3′ 681NM_007614.2 Mm.291928 Catnb Catenin beta 5′-CUGUUGUGGUUAAACUCCUTT-3′ 682NM_011232 Mm.38376 Rad1 RAD1 homolog (S. pombe)5′-GCCTTGACAACGTTAGGAATC-3′ 683 NM_011232 Mm.38376 Rad1RAD1 homolog (S. pombe) 5′-GCAGGAAGTTCCCACCTTGAC-3′ 684 NM_011232Mm.38376 Rad1 RAD1 homolog (S. pombe) 5′-GCCTGATGAAGAAGTTCC-3′ 685NM_010423 Mm.29581 Hcy1 Hairy/enhancer-of-split 5′-GCTAGAAAAAGCTGAGATC-3′ 686 related with YRPW motif 1 NM_010133Mm.2657 En1 Engrailed 1 5′-CAUCCUAAGGCCCGAUUUCTT-3′ 687 NM_010133Mm.2657 En1 Engrailed 1 5′-GUUCCCGGAACACAACCCUTT-3′ 688 NM_019390Mm.243014 Lmna Lamin A 5′-GCAGCUUCAGGAUGAGAUGTT-3′ 689 NM_183355.1Mm.43358 Pbx1 Pre B-cell leukemia 5′-CAGUUUUGAGUAUUCGGGGTT-3′ 690transcription factor 1 NM_007430 Mm.5180 Nr0b1Nuclear receptor subfamily 0, 5′-GAUCACCUGCACUUCGAGdTdT-3′ 691group B, member 1 NM_007430 Mm.5180 Nr0b1 Nuclear receptor subfamily 0,5′-CUGAACAGUGCCCUUUUCCdTdT-3′ 692 group B, member 1 NM_172203 Mm.233865Nox1 NADPH oxidase 1 5′-TTATGAGAAGTCTGACAAG-3′ 693 NM_172203 Mm.233865Nox1 NADPH oxidase 1 5′-GATTCTTGGCTAAATCCCA-3′ 694 NM_172203 Mm.233865Nox1 NADPH oxidase 1 5′-GGACATTTGAACAACAGCA-3′ 695 NM_080850 Mm.253287Pask PAS domain containing 5′-AATTTATGGAGTCAACCACAGCTT-3′ 696serine/threonine kinase NM_009330 Mm.7226 Tcf2 Transcription factor 25′-GCCGGUUUUCCAUACUCUCtt-3′ 697 NM_009330 Mm.7226 Tcf2Transcription factor 2 5′-CAAGAAGAUGCGCCGCAACtt-3′ 698 NM_009330 Mm.7226Tcf2 Transcription factor 2 5′-UGGUGGUCACAGAUACCAGtt-3′ 699 NM_024148.1Rn.5949 Apex1 Apurinic/apyrimidinic 5′-GUCUGGUAAGACUGGAGUACC-3′ 700endonuclease 1 NM_017059.1 Rn.10668 Bax Bcl2-associated X protein5′-UUGGAGAUGAACUGGACAAUU-3′ 701 NM_017059.1 Rn.10668 BaxBcl2-associated X protein 5′-CUG GAC AAU AAU AUG GAG  702 CUU-3′NM_023979.1 Rn.64522 Apaf1 Apoptotic protease activating5′-AGA ACU UUG UGC UUU AAU  703 factor 1 GUU-3′ NM_023979.1 Rn.64522Apaf1 Apoptotic protease activating 5′-UAU AGG CAU AUA CUG GAU  704factor 1 GUU-3′ NM_021835.2 Rn.93714 Jun V-jun sarcoma virus 175′-AGU GAA AAC CUU GAA AGC  705 oncogene homolog (avian) GUU-3′NM_021835.2 Rn.93714 Jun V-jun sarcoma virus 175′-AGU CAU GAA CCA CGU UAA  706 oncogene homolog (avian) CUU-3′NM_012655.1 Rn.44609 Sp1 Sp1 transcription factor AATGAGAACAGCAACAACTCC707 XM_230974.2 Rn.102138 Sp3 Sp3 transcription factorAAGTTCTCAGACAATGACTGC 708 NM_133551.1 Rn.10162 Pla2g4aPhospholipase A2, group IVA 5′-TCGAGACAGTAGTGGTTCTACGTG 709(cytosolic, calcium-dependent) CCgagtactgGGCACGTAGAACCACTA CTGTTTTTT-3′NM_001003959.1 Rn.117353 Dnmt3b DNA methyltransferase 3B5′-AGAUGACAGGUGCCCAGAGUU-3′ 710 NM_019335.1 Rn.10022 PrkrProtein kinase, interferon- 5′-GGUAGAUCAAAGCAGGAGGTT-3′ 711inducible double stranded RNA  dependent NM_053622.1 Rn.10474 Pom121Nuclear pore membrane 5′-AACGGAGUCCCUGCUGCAUUU 712 glycoprotein 121 kDdTdT-3′ NM_053622.1 Rn.10474 Pom121 Nuclear pore membrane5′-AACCAUGUCACCAGUCCAGUU 713 glycoprotein 121 kD dTdT-3′ NM_053622.1Rn.10474 Pom121 Nuclear pore membrane 5′-AAGCCUGUGUUUGGCUUUGGA 714glycoprotein 121 kD dTdT-3′ NM_131907.2 Rn.5805 Atp2c1ATPase, Ca++-sequestering 5′-AACCATTATGGAAGAAGTACATT-3′ 715 NM_031337.1Rn.22706 Siar9 Sialyltransferase 9 5′-GGGUUAUUCUGAACAUGUUtt-3′ 716NM_031010.2 Rn.11318 Alox15 Arachidonate 12-lipoxygenase5′-GCAACTGGATTTCTGTGAAGG-3′ 717 NM_031010.2 Rn.11318 Alox15Arachidonate 12-lipoxygenase 5′-GAAGCGGATTTCTTCCTTCTG-3′ 718 NM_031556.1Rn.22518 Cav Caveolin 5′-AAGGAGATCGACCTGGTCAAC-3′ 719 NM_031556.1Rn.22518 Cav Caveolin 5′-AAGGGACACACAGTTTTGACG-3′ 720 XM_232106.2Rn.117974 Dok-1 Docking protein 1 (predicted)5′-GAACTACACAAATTCAGCCAGGCG 721 TATCATCCGGTGTTTCGTCCTTTCCAC AAG-3′NM_133307.1 Rn.98279 Prkcd Protein kinase C, delta5′-AAAAGGCAAATTCACAAACAGCCT 722 GTCTC-3′ NM_133307.1 Rn.98279 PrkcdProtein kinase C, delta 5′-AAGTTCTCCGAAGTGTGAGAACCT 723 GTCTC-3′NM_012637.1 Rn.11317 Ptpn1 Protein tyrosine phosphatase,5′-AAGCTGACACTGATCTCTGAA-3′ 724 non-receptor type 1 NM_017212 Rn.2455Mapt Microtubule-associated protein 5′-CCAGGAGTTTGACACAATG-3′ 725 tauNM_013156 Rn.1294 CTSL Cathepsin L 5′-GGACAGAUGUUCCUUAAGATT-3′ 726NM_012576.1 Rn.90070 Nr3c1 Nuclear receptor subfamily 3,5′-GGCCAAGGGAGGGGGAGCGTA-3′ 727 group C, member 1 NM_013131.1 Rn.9678Nr3c2 Nuclear receptor subfamily 3, 5′-GGCGCTGGAGTCAAGTGTCTC-3′ 728group C, member 2 NM_031659 Rn.10039 Tgm1 Transglutaminase 15′-AUGCAGCUGGAGAUGGCACdTdT-3′ 729 NM_019275 Rn.9774 Madh4 MAD homolog 45′-AAUACACCGACAAGCAAUGACdTdT-3′ 730 NM_031132 Rn.9954 Tgfbr2Transforming growth factor, 5′-AAAGUCGGUUAACAGCGAUCUdTdT-3′ 731beta receptor II NM_144741 Rn.16746 Retn Resistin5′-CCTTTCATTTCTCCTCCTT-3′ 732 NM_144741 Rn.16746 Retn Resistin5′-AGCTGCTCCTGTGGCTCTG-3′ 733 NM_144741 Rn.16746 Retn Resistin5′-GCCTCCTGCCCAGAAGGCA-3′ 734 NM_012614 Rn.9714 Npy Neuropeptide Y5′-UGAGAGAAAGCACAGAAA-3′ 735 NM_021597 Rn.35512 Eif2c2 GERp955′-UGGACAUCCCCAAAAUUGA-3′ 736 NM_012547 Rn.87299 Drd2Dopamine receptor 2 5′-CCCCAUCAUCUACACCACA-3′ 737

APPENDIX (B)

U.S. Pat. No. 6,207,646 B1

TABLE 1 Oligonucleotide Stimulation of Mouse B Cells ODNStimulation Index′  Production Sequence (5′ to 3′) ³H Uridine IgM 1(SEQ ID NO: 1) GCTAGACGTTAGCGT  6.1 ± 0.8 17.9 ± 3.6  1a (SEQ ID NO: 2)......T........  1.2 ± 0.2  1.7 ± 0.5 1b (SEQ ID NO: 3) ......Z........ 1.2 ± 0.1  1.8 ± 0.0 1c (SEQ ID NO: 4) ............Z.. 10.3 ± 4.4  9.5 ± 1.8 1d (SEQ ID NO: 5) ..AT.......GAGC. 13.0 ± 2.3  18.3 ± 7.5  2(SEQ ID NO: 6) ATGGAAGGTCCAGCGTTCTC  2.9 ± 0.2 13.6 ± 2.0  2a(SEQ ID NO: 7) ..C..CTC..G.........  7.7 ± 0.8 24.2 ± 3.2  2b(SEQ ID NO: 8) ..Z..CTC.ZG..Z......  1.6 ± 0.5  2.8 ± 2.2 2c(SEQ ID NO: 9) ..Z..CTC..G.........  3.1 ± 0.6  7.3 ± 1.4 2d(SEQ ID NO: 10) ..C..CTC..G......Z..  7.4 ± 1.4 27.7 ± 5.4  2e(SEQ ID NO: 11) ............A.......  5.6 ± 2.0 ND 3D (SEQ ID NO: 12)GAGAACGCTGGACCTTCCAT  4.9 ± 0.5 19.9 ± 3.6  3Da (SEQ ID NO: 13).........C..........  6.6 ± 1.5 33.9 ± 6.8  3Db (SEQ ID NO: 14).........C.......G.. 10.1 ± 2.8  25.4 ± 0.8  3Dc (SEQ ID NO: 15)...C.A..............  1.0 ± 0.1  1.2 ± 0.5 3Dd (SEQ ID NO: 17).....Z..............  1.2 ± 0.2  1.0 ± 0.4 3De (SEQ ID NO: 12).............Z......  4.4 ± 1.2 18.8 ± 4.4  3Df (SEQ ID NO: 18).......A............  1.6 ± 0.1  7.7 ± 0.4 3Dg (SEQ ID NO: 19).........CC.G.ACTG..  6.1 ± 1.5 18.6 ± 1.5  3M (SEQ ID NO: 22)TCCATGTCGGTCCTGATGCT  4.1 ± 0.2 23.2 ± 4.9  3Ma (SEQ ID NO: 21)......CT............  0.9 ± 0.1  1.8 ± 0.5 3Mb (SEQ ID NO: 22).......Z............  1.3 ± 0.3  1.5 ± 0.6 3Mc (SEQ ID NO: 23)...........Z........  5.4 ± 1.5  8.5 ± 2.6 3Md (SEQ ID NO: 24)......A..T.......... 17.2 ± 9.4  ND 3Me (SEQ ID NO: 741)...............C..A.  3.6 ± 0.2 14.2 ± 5.2  4 TCAACGTT (SEQ ID NO: 6.1 ± 1.4 19.2 ± 5.2  26) 4a ....GC.. (SEQ ID NO:  1.1 ± 0.2  1.5 ± 1.127) 4b ...GCGC. (SEQ ID NO:  4.5 ± 0.2  9.6 ± 3.4 28) 4c...TCGA. (SEQ ID NO:  2.7 ± 1.0 ND 2306) 4d ..TT..AA (SEQ ID NO: 1.3 ± 0.2 ND 30) 4e ........ (SEQ ID NO:  1.3 ± 0.2 1.1 ± 0.5 31) 4fC....... (SEQ ID NO:  3.9 ± 1.4 ND 32) 4g --......CT (SEQ ID  1.4 ± 0.3ND NO: 2307) 4h .......C (SEQ ID NO:  1.2 ± 0.2 ND 34) LPS  7.8 ± 2.84.8 ± 1.0 ′Stimulation indexes are the means and std. dev. derived fromat least 3 separate experiments, and are compared to wells cultured withno added ODN. ND = not done. CpG dinucleotides are underlined. Dotsindicate identity; dashes indicate deletion. Z indicates 5 methylcytonine.

TABLE 2Identification of the optimal CpG motif for Nurine IL-6 production and B cell activation.IL-6 (pg/ml)^(a) ODN SPLENIC (ng/ml)^(C) SEQUENCE (5′-3′) CH12.LX B CELLSI^(b) IgM 512 (SEQ ID No: 22) TCCATGTCGGTCCTGATGCT 1300 ± 106  627 ± 43 5.8 ± 0.3  7315 ± 1324 1637 (SEQ ID No: 43) ......C............. 136 ± 27   46 ± 6  1.7 ± 0.2   770 ± 72 1615 (SEQ ID No: 44)......G............. 1201 ± 155  850 ± 202  3.7 ± 0.3  3212 ± 617 1614(SEQ ID No: 45) ......A............. 1533 ± 321 1812 ± 103 10.8 ± 0.6  7558 ± 414 1636 (SEQ ID No: 46) .........A.......... 1181 ± 76  947 ± 132  5.4 ± 0.4  3983 ± 485 1634 (SEQ ID No: 47).........C.......... 1049 ± 223 1671 ± 175  9.2 ± 0.9  6256 ± 261 1619(SEQ ID No: 48) .........T.......... 1555 ± 304 2908 ± 129 12.5 ± 1.0  8243 ± 698 1618 (SEQ ID No: 24) ......A..T.......... 2109 ± 2912596 ± 166 12.9 ± 0.7  10425 ± 674  1639 (SEQ ID No: 49).....AA..T.......... 1827 ± 83  2012 ± 132 11.5 ± 0.4   9439 ± 103 1707(SEQ ID No: 50) ......A..TC......... ND 1147 ± 175  4.0 ± 0.2 3534 ± 217 1708 (SEQ ID No: 51) .....CA..TG......... ND 59 ± 3 1.5 ± 0.1   466 ± 109 Dots indicate identity; CpG dinucleotides areunderlined; ND = not done. The experiment was done at least three timeswith similar results. The level of IL-6 of unstimulated control culturesof both CH.2.LX and splenic B cells was ≤ 10 pg/ml. The IgM level ofunstimulated culture was 547 ± 82 ng/ml. CpG dinucleotides areunderlined and dots indicate identity. ^(b)[³H] Uridine uptake wasindicated as a fold increase (SI: stimulation index) from unstimulatedcontrol (2322.67 ± 213.68 cpm). Cells were stimulated with 20 μM ofvarious CpG O-QDN. Data present the mean ± SD of triplicates Measured byELISA.

TABLE 3 Induction of Murine IL-6 secretion by CpG motifsin bacterial DNA or oligonucleotides. Treatment IL-6 (pg/ml)calf thymus DNA ≤10 calf thymus DNA + DNase ≤10 E. coli DNA1169.5 ± 94.1  E. coli DNA + DNase ≤10 CpG methylated E. coli DNA ≤10LPS 280.1 ± 17.1 Media (no DNA) ≤10 ODN 5a SEQ. ATGGACTCTCCAGCGTTCTC1096.4 ± 372.0 ID No: 35 5b SEQ. .....AGG....A....... 1124.5 ± 126.2 IDNo:  740 5c SEQ. ..C.......G......... 1783.0 ± 189.5 ID No: 7 5d SEQ..... AGG..C..T...... ≤10 ID No:  4742 5e SEQ. ..C.......G..Z...... 851.1 ± 114.4 ID No: 8 5f SEQ. ..Z......ZG..Z...... ≤10 ID No: 9 5gSEQ. ..C.......G......Z.. 1862.3 ± 87.26 ID No: 10

TABLE 5 Induction of human PBMC cytokine secrtetion by CpG oligos ODNSequence (5′-3′) IL-6¹ TNF-α¹ IFN-γ¹ GM-CSF IL-12 512TCCATGTCGGTCCTGATGCT 500 140 15.6 70 250 SEQ ID NO: 22 1637......C............. 550 16 7.8 15.6 35 SEQ ID NO: 43 1615......G............. 600 145 7.8 45 250 SEQ ID NO: 44 1645......A............. 550 31 0 50 250 (SEQ ID NO: 45) 1636.........A.......... 325 250 35 40 0 SEQ ID NO: 46 1634.........C.......... 300 400 40 85 200 SEQ ID NO: 47 1619.........T.......... 275 450 200 80 ≥500 SEQ ID NO: 48 1618......A..T.......... 300 60 15.6 15.6 62 SEQ ID NO: 24 1639.....AA..T.......... 625 220 15.6 40 60 SEQ ID NO: 49 1707......A..TC......... 300 70 17 0 0 SEQ ID NO: 50 1708.....CA..TG......... 270 10 17 0 0 SEQ ID NO: 51

TABLE 9 Induction of NK Activity by DNA Containing CpG Motifs but notby Non-CpG DNA LU/10⁶ DNA or Cytokine Added Mouse Cells Human cellsExpt. 1 None 0.00 0.00 IL-2 16.68 15.82 E. Coli, DNA 7.23 5.05Calf thymus DNA 0.00 0.00 Expt. 2 None 0.00 3.281585 ggGGGGTCAACGTTGAGGGGGGgggg (SEQ ID No. 743) 7.38 17.981629 ----------gtc------------- (SEQ ID No. 744) 0.00 4.4 Expt. 3 None0.00 1613 GCTAGACGTTAGTGT (SEQ ID No. 54) 5.22 1769 -------Z-------(SEQ ID No. 745) 0.02 ND 1619 TCCATGTCGTTCCTGATGCT (SEQ ID No: 48) 3.351765 -------Z------------ (SEQ ID No. 56) 0.11

TABLE 10 Different CpG motifs stimulate optimalmurine B cell and NK activation B cell NK ODM Sequence activation¹activation² 1668 TCCATGACGTTCCTGATGCT 42,849 2.52 (SEQ. ID. NO: 24) 1758TCTCCCAGCGTGCGCCAT 1,747 6.66 (SEQ. ID. NO. 59) NONE 367 0.00 CpGdinucleotides are underlined; oligonucleotides were synthesized withphosphorothioate modified backbones to improve their nucleaseresistance. ¹Measured by ³H thymidine incorporation after 48 hr culturewith oligodeoxynucleotides at a 200 nM concentration as described inExample 1. ²Measured in lytic units.U.S. Pat. No. 6,214,806 B1

TABLE 1 ODN Number cells/ml × 10⁴ PMN/ml × 10³ % PMN expt 1, C3H/BFeJmice 1908 61.4 ± 15.9 59.2 ± 15.7 95.8 ± 0.95 1760 27.8 ± 3.5* 25.8 ±3.0* 93.3 ± 2.3 1631 47.6 ± 11.1 46.1 ± 10.7 96.8 ± 1.11 1835 43.8 ± 7.144.4 ± 7.1 96.8 ± 0.75 1759 71.0 ± 19.8 67.7 ± 20.4 96.8 ± 2.6 1826 39.8± 7.8 38.3 ± 7.9 93.5 ± 1.4 None (saline) 71.0 ± 7.4 69.3 ± 6.9 97.8 ±1.3 expt 2, C57 Bl/6 mice 1908 18.0 ± 2.6 16.6 ± 2.7 91.2 ± 3.7 176010.2 ± 2.3*  8.6 ± 2.1* 82.0 ± 3.0 1585 11.0 ± 2.2*  9.5 ± 2.2* 84.6 ±2.9 2010 14.1 ± 2.1 11.8 ± 1.9 83.4 ± 2.1 None (saline) 17.9 ± 3.4 16.9± 2.1 expt 3 1908 19.0 ± 2.5 16.9 ± 2.1 89.4 ± 1.4 1760  9.1 ± 0.8*  7.7± 0.7* 84.6 ± 0.5 1972 15.3 ± 1.6 13.5 ± 1.4 84.2 ± 1.2 2001 13.0 ± 1.6*11.8 ± 1.6 90.4 ± 2.2 *P > 0.05, Mean-Whitney U test

TABLE 2 Induction Of NK Activity By CpG Oligodeoxynucleotides (ODN) %YAC-1 Specific Lysis* % 2C11 Specific Lysis Effector:TargetEffector:Target ODN 50:1 100:1 50:1 100:1 None −1.1 −1.4 15.3 16.6 116.1 24.5 38.7 47.2 3Dd 17.1 27.0 37.0 40.0 non-CpG ODN −1.6 −1.7 14.815.4

TABLE 3 Induction of NK Activity by DNAContaining CpG Motifs but not by Non-CpG DNA LU/10⁵ Mouse HumanDNA or Cytokine Added Cells Cells Expt. 1 None 0.00 0.00 IL-2 16.6815.82 E. Coli DNA 7.23 5.05 Calf thymus DNA 0.00 0.00 Expt. 2 None 0.003.28 1585 ggGGTCAACGTTGACgggg 7.38 17.98 (SEQ ID NO: 746)1629 --------gtc-------- 0.00 4.4 (SEQ ID NO: 747) Expt. 3 None 0.001613 GCTAGACGTTAGTGT 5.22 (SEQ ID NO: 54) 1769 -------X----- 0.02 ND(SEQ ID NO: 745) 1619 TCCATGTCGTTCCTGATGCT 3.35 (SEQ ID NO: 48)1765 --------X----------- 0.11 (SEQ ID NO: 748) CpG dinucleotides in ODNsequences are indicated by underlining; X indicates methylcytosine.Lower case letters indicate nuclease resistant phosphorothioate modifiedinternucleotide linkages which, in titration experiments, were more than20 times as potent as non-modified ODN, depending on the flanking bases.Poly G ends (g) were used in some ODN, because they significantlyincrease the level of ODN uptake. Dashes indicate some bases areidentical to those in the directly preceding sequence, with theexception of changes noted.

TABLE 4 ODN induction of NK Lytic Activity (LU) ODN Sequence (5′-3′) LUNone 0.01 1754 ACCATGGACGATCTGTTTCCCCTC 0.02 (SEQ ID NO: 58) 1758TCTCCCAGCGTGCGCCAT 0.05 (SEQ ID NO: 59) 1761 TACCGCGTGCGACCCTCT 0.05(SEQ ID NO: 60) 1776 ACCATGGACGAACTGTTTCCCCTC 0.03 (SEQ ID NO: 61) 1777ACCATGGACGAGCTGTTTCCCCTC 0.05 (SEQ ID NO: 62) 1778ACCATGGACGACCTGTTTCCCCTC 0.01 (SEQ ID NO: 63) 1779ACCATGGACGTACTGTTTCCCCTC 0.02 (SEQ ID NO: 64) 1780ACCATGGACGGTCTGTTTCCCCTC 0.29 (SEQ ID NO: 65) 1781ACCATGGACGTTCTGTTTCCCCTC 0.38 (SEQ ID NO: 66) 1823 GCATGACGTTGAGCT 0.08(SEQ ID NO: 41) 1824 CACGTTGAGGGGCAT 0.01 (SEQ ID NO: 67) 1825CTGCTGAGACTGGAG 0.01 (SEQ ID NO: 68) 1828 TCAGCGTGCGCC 0.01(SEQ ID NO: 69) 1829 ATGACGTTCCTGACGTT 0.42 (SEQ ID NO: 70) 1830₂RANDOM SEQUENCE 0.25 1834 TCTCCCAGCGGGCGCAT 0.00 (SEQ ID NO: 71) 1836TCTCCCAGCGCGCGCCAT 0.46 (SEQ ID NO: 72) 1840 TCCATGTCGTTCCTGTCGTT 2.70(SEQ ID NO: 73) 1841 TCCATAGCGTTCCTAGCGTT 1.45 (SEQ ID NO: 74) 1842TCGTCGCTGTCTCCGCTTCTT 0.06 (SEQ ID NO: 75) 1851 TCCTGACGTTCCTGACGTT 2.32(SEQ ID NO: 76) ₁Lytic units (LU) were measured as described (8).Briefly, PBMC were collected from normal donors and spun over Ficoll,then cultured with or without the indicated ODN (which were added tocultures at 6 μg/ml) for 24 hr. Then their ability to lyse ⁵¹Cr-labeledK562 cells was determined. The results shown are typical of thoseobtained with several different normal human donors. ₂This oligo mixturecontained a random selection of all 4 bases at each position.

TABLE 5 Induction of NK LU by Phoshorothioate Cp GODN with Good MotifsODN₁ sequence (5′-3′) expt. 1 expt. 2 expt. 3 None 0.00 1.26 0.46 1840TCCATGTCGTTCCTGTCGTT (SEQ ID NO: 73) 2.33 ND ND 1960TCCTGTCGTTCCTGTCGTT (SEQ ID NO: 77) ND 0.48 8.99 1961TCCATGTCGTTTTTGTCGTT (SEQ ID NO: 78) 4.03 1.23 5.08 1962TCCTGTCGTTCCTTGTCGTT (SEQ ID NO: 79) ND 1.60 5.74 1963TCCTTGTCGTTCCTGTCGTT (SEQ ID NO: 80) 3.42 ND ND 1965TCCTGTCGTTTTTTGTCGTT (SEQ ID NO: 81) 0.46 0.42 3.48 1966TCGTCGCTGTCTCCGCTTCTT (SEQ ID NO: 75) 2.62 ND ND 1967TCGTCGCTGTCTGCCCTTCTT (SEQ ID NO: 82) 5.82 1.64 8.32 1968TCGTCGCTGTTGTCGTTTCTT (SEQ ID NO: 83) 3.77 5.26 6.12 1979₂TCCATGTZGTTCCTGTZGTT (SEQ ID NO: 84) 1.32 ND ND 1982TCCAGGACTTCTCTCAGGTT (SEQ ID NO: 85) 0.05 ND 0.98 1990TCCATGCGTGCGTGCGTTTT (SEQ ID NO: 86) 2.10 ND ND 1991TCCATGCGTTGCGTTGCGTT (SEQ ID NO: 87) 0.89 ND ND 2002TCCACGACGTTTTCGACGTT (SEQ ID NO: 88) 4.02 1.31 9.79 2005TCGTCGTTGTCGTTGTCGTT (SEQ ID NO: 89) ND 4.22 12.75 2006TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 90) ND 6.17 12.82 2007TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO: 91) ND 2.68 9.66 2008GCGTGCGTTGTCGTTGTCGTT (SEQ ID NO: 92) ND 1.37 8.15 2010GCGGCGGGCGGCGCGCGCCC (SEQ ID NO: 93) ND 0.01 0.05 2012TGTCGTTTGTCGTTTGTCGTT (SEQ ID NO: 94) ND 2.02 11.61 2013TGTCGTTGTCGTTGTCGTTGTCGTT (SEQ ID NO: 95) ND 0.56 5.22 2014TGTCGTTGTCGTTGTCGTT (SEQ ID NO: 96) ND 5.74 10.89 2015TCGTCGTCGTCGTT (SEQ ID NO: 97) ND 4.53 10.13 2016TGTCGTTGTCGTT (SEQ ID NO: 98) ND 6.54 8.06 ₁PBMC essentially asdescribed herein. Results are representative of 6 separate experiments;each experiment represents a different donor. ₂This is the methylatedversion of ODN 1840; Z = 5-methyl cytosine LU is lytic units; ND = notdone; CpG dinucleotides are underlined for clarity.

TABLE 6Induction of human B cell proliferation by Phosphorothioate CpG ODNStimulation Index¹ expt. DB sequence (5′-3′) 1 expt. 2 expt. 3 expt. 4expt. 5 expt. 6 1840 TCCATGTCGTTCCTGTCGTT (SEQ ID NO: 84) 4 ND ND ND ND34 1841 TCCATAGCGTTCCTAGCGTT (SEQ ID NO: 99) 3 ND ND ND ND ND 1960TCCTGTCGTTCCTGTCGTT (SEQ ID NO: 77) ND 2.0 2.0 3.6 ND ND 1961TCCATGTCGTTTTTGTCGTT (SEQ ID NO: 78) 2 3.9 1.9 3.7 ND 37 1962TCCTGTCGTTCCTTGTCGTT (SEQ ID NO: 79) ND 3.8 1.9 3.9 5.4 35 1963TCCTTGTCGTTCCTGTCGTT (SEQ ID NO: 80) 3 ND ND ND ND ND 1965TCCTGTCGTTTTTTGTCGTT (SEQ ID NO: 81) 4 3.7 2.4 4.7 6.0 43 1967TCGTCGCTGTCTGCCCTTCTT (SEQ ID NO: 82) ND 4.4 2.0 4.5 5.0 36 1968TCGTCGCTGTTGTCGTTTCTT (SEQ ID NO: 83) ND 4.0 2.0 4.9 8.7 38 1982TCCAGGACTTCTCTCAGGTT (SEQ ID NO: 85) 3 1.8 1.3 3.1 3.2 12 2002TCCACGACGTTTTCGACGTT (SEQ ID NO: 88) ND 2.7 1.4 4.4 ND 14 2005TCGTCGTTGTCGTTGTCGTT (SEQ ID NO: 89) 5 3.2 1.2 3.0 7.9 37 2006TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 90) 4 4.5 2.2 5.8 8.3 40 2007TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO: 91) 3 4.0 4.2 4.1 ND 22 2008GCGTGCGTTGTCGTTGTCGTT (SEQ ID NO: 92) ND 3.0 2.4 1.6 ND 12 2010GCGGCGGGCGGCGCGCGCCC (SEQ ID NO: 93) ND 1.6 1.9 3.2 ND ND 2012TGTCGTTTGTCGTTTGTCGTT (SEQ ID NO: 94) 2 2.8 0   3.2 ND 33 2013TGTCGTTGTCGTTGTCGTTGTCGTT (SEQ ID NO: 95) 3 2.3 3.1 2.8 ND  7 2014TGTCGTTGTCGTTGTCGTT (SEQ ID NO: 96) 3 2.5 4.0 3.2 6.7 14 2015TCGTCGTCGTCGTT (SEQ ID NO: 97) 5 1.8 2.6 4.5 9.4  1 2016TGTCGTTGTCGTT (SEQ ID NO: 98) ND 1.1 1.7 2.7 7.3  1 ¹Cells = humanspleen cells stored at −70° C. after surgical harvest or PBMC collectedfrom normal donors and spun over Ficoll. Cells were cultured in 96 wellU-bottom microtiter plates with or without the indicated ODN (which wereadded to cultures at 6 μml). N = 12 experiments. Cells were cultured for4-7 days, pulsed with 1 μCi of ³H thymidine for 18 hr before harvest andscintillation counting. Stimulation index = the ratio of cpm in wellswithout ODN to that in wells that had been stimulated throughout theculture period with the indicated ODN (there were no further additionsof ODN after the cultures were set up). ND = not done

TABLE 7 Induction of human IL-12 secretion by Phosphorothioate CpG ODNIL-12 (pg/ml) ODN₁ sequence (5′-3′) expt. 1 expt. 2 None 0 0 1962TCCTGTCGTTCCTTGTCGTT 19 0 (SEQ ID NO: 79) 1965 TCCTGTCGTTTTTTGTCGTT 36 0(SEQ ID NO: 81) 1967 TCGTCGCTGTCTGCCCTTCTT 41 0 (SEQ ID NO: 82) 1968TCGTCGCTGTTGTCGTTTCTT 24 0 (SEQ ID NO: 83) 2005 TCGTCGTTGTCGTTGTCGTT 250 (SEQ ID NO: 89) 2006 TCGTCGTTTTGTCGTTTTGTCGTT 29 15 (SEQ ID NO: 90)2014 TGTCGTTGTCGTTGTCGTT 28 0 (SEQ ID NO: 96) 2015 TCGTCGTCGTCGTT 14 0(SEQ ID NO: 97) 2016 TGTCGTTGTCGTT 3 0 (SEQ ID NO: 98) ₁PBMC werecollected from normal donors and spun over Ficoll, then cultured at 10⁶cells/well in 96 well microtiter plates with or without the indicatedODN which were added to cultures at 6 μg/ml. Supernatants were collectedat 24 hr and tested for IL-12 levels by ELISA as described in methods. Astandard curve was run in each experiment, which represents a differentdonor.

TABLE 8 Different CpG motifs stimulate optimal murineB cell and NK activation B cell NK ODN Sequence activation¹ activation²1668 TCCATGACGTTCCTGATGCT 42,849 2.52 (SEQ ID NO: 24) 1758TCTCCCAGCGTGCGCCAT 1,747 6.66 (SEQ ID NO: 59) NONE 367 0.00 CpGdinucleotides are underlined; oligonucleotides were synthesized withphosphorothioate modified backbones to improve their nucleaseresistance. ¹Measured by ³H thymidine incorporation after 48 hr culturewith oligodeoxy-nucleotides at a 200 nM concentration as described inExample 1. ²Measured in lytic units.U.S. Pat. No. 6,218,371 B1

TABLE 1 sequences GCTAGACGTTAGCGT (SEQ ID NO: 1) GCTAGATGTTAGCGT(SEQ ID NO: 2) GCTAGAZGTTAGCGT (SEQ ID NO: 3) GCTAGACGTTAGZGT(SEQ ID NO: 4) GCATGACGTTGAGCT (SEQ ID NO: 5) ATGGAAGGTCCAGCGTTCTC(SEQ ID NO: 6) ATCGACTCTCGAGCGTTCTC (SEQ ID NO: 7) ATZGACTCTZGAGZGTTCTC(SEQ ID NO: 8) ATZGACTCTCGAGCGTTCTC (SEQ ID NO: 9) ATCGACTCTCGAGCGTTZTC(SEQ ID NO: 10) ATCGACTCTCGAACGTTCTC (SEQ ID NO: 11)GAGAACGCTGGACCTTCCAT (SEQ ID NO: 12) GAGAACGCTCGACCTTCCAT(SEQ ID NO: 13) GAGAACGCTCGACCTTCGAT (SEQ ID NO: 14)GAGCAAGCTGGACCTTCCAT (SEQ ID NO: 15) GAGCAZGCTGGACCTTCCAT(SEQ ID NO: 16) GAGAACGCTGGACZTTCCAT (SEQ ID NO: 17)GAGAACGATGGACCTTCCAT (SEQ ID NO: 18) TCCATGTZGTTCCTGATGCT(SEQ ID NO: 57) ACCATGGACGATCTGTTTCCCCTC (SEQ ID NO: 58)TCTCCCAGCGTGCGCCAT (SEQ ID NO: 59) TACCGCGTGCGACCCTCT (SEQ ID NO: 60)ACCATGGACGAACTGTTTCCCCTC (SEQ ID NO: 61) ACCATGGACGAGCTGTTTCCCCTC(SEQ ID NO: 62) ACCATGGACGACCTGTTTCCCCTC (SEQ ID NO: 63)ACCATGGACGTACTGTTTCCCCTC (SEQ ID NO: 64) ACCATGGACGGTCTGTTTCCCCTC(SEQ ID NO: 65) ACCATGGACGTTCTGTTTCCCCTC (SEQ ID NO: 66) CACGTTGAGGGGCAT(SEQ ID NO: 67) CTGCTGAGACTGGAG (SEQ ID NO: 68) TCAGCGTGCGCC(SEQ ID NO: 69) ATGACGTTCCTGACGTT (SEQ ID NO: 70) TCTCCCAGCGGGCGCAT(SEQ ID NO: 71) TCTCCCAGCGCGCGCCAT (SEQ ID NO: 72) TCCATGTCGTTCCTGTCGTT(SEQ ID NO: 73) TCCATAGCGTTCCTAGCGTT (SEQ ID NO: 74)TCGTCGCTGTCTCCGCTTCTT (SEQ ID NO: 75) TCCTGACGTTCCTGACGTT(SEQ ID NO: 76) TCCTGTCGTTCCTGTCGTT (SEQ ID NO: 77) TCCATGTCGTTTTTGTCGTT(SEQ ID NO: 78) TCCTGTCGTTCCTTGTCGTT (SEQ ID NO: 79)TCCTTGTCGTTCCTGTCGTT (SEQ ID NO: 80) TCCTGTCGTTTTTTGTCGTT(SEQ ID NO: 81) TCGTCGCTGTCTGCCCTTCTT (SEQ ID NO: 82)TCGTCGCTGTTGTCGTTTCTT (SEQ ID NO: 83) TCCATGTZGTTCCTGTZGTT(SEQ ID NO: 84) TCCAGGACTTCTCTCAGGTT (SEQ ID NO: 85)TCCATGCGTGCGTGCGTTTT (SEQ ID NO: 86) TCCATGCGTTGCGTTGCGTT(SEQ ID NO: 87) TCCACGACGTTTTCGACGTT (SEQ ID NO: 88)TCGTCGTTGTCGTTGTCGTT (SEQ ID NO: 89) TCGTCGTTTTGTCGTTTTGTCGTT(SEQ ID NO: 90) TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO: 91)GCGTGCGTTGTCGTTGTCGTT (SEQ ID NO: 92) GCGGCGGGCGGCGCGCGCCC(SEQ ID NO: 93) TGTCGTTTGTCGTTTGTCGTT (SEQ ID NO: 94)TGTCGTTGTCGTTGTCGTTGTCGTT (SEQ ID NO: 95) TGTCGTTGTCGTTGTCGTT(SEQ ID NO: 96) TCGTCGTCGTCGTT (SEQ ID NO: 97) TGTCGTTGTCGTT(SEQ ID NO: 98) TCCATAGCGTTCCTAGCGTT (SEQ ID NO: 99)TCCATGACGTTCCTGACGTT (SEQ ID NO: 100) GTCG(T/C)T (SEQ ID NO: 101)TGTCG(T/C)T (SEQ ID NO: 102) TCCATGAGCTTCCTGAGTCT (SEQ ID NO: 103)TCTCCCAGCGTGCGCCAT (SEQ ID NO: 104) TCCATGACGTTCCTGACGTT(SEQ ID NO: 105)

TABLE 2 Induction of human IL-12 secretion byPhosphorothioate CpG oliqonucleotide IL-12 (pg/ml) ODN₁ sequence (5′-3′)expt. 1 expt. 2 None 0 0 1962 TCCTGTCGTTCCTTGTCGTT 19 0 (SEQ. ID NO: 79)1965 TCCTGTCGTTTTTTGTCGTT 36 0 (SEQ. ID NO: 81) 1967TCGTCGCTGTCTGCCCTTCTT 41 0 (SEQ. ID NO: 82) 1968 TCGTCGCTGTTGTCGTTTCTT24 0 (SEQ. ID NO: 83) 2005 TCGTCGTTGTCGTTGTCGTT 25 0 (SEQ. ID NO: 89)2006 TCGTCGTTTTGTCGTTTTGTCGTT 29 15 (SEQ ID NO: 90) 2014TGTCGTTGTCGTTGTCGTT 28 0 (SEQ. ID NO: 96) 2015 TCGTCGTCGTCGTT 14 0(SEQ ID NO: 97) 2016 TGTCGTTGTCGTT 3 0 (SEQ. ID NO: 98) ₁PBMC werecollected from normal donors and spun over Ficoll, then cultured at 10⁶cells/well in 96 well microtiter plates with or without the indicatedoligonucleotide which were added to cultures at μg/ml. Supernatants werecollected at 24 hr and tested for IL-12 levels by ELISA as described inmethods. A standard curve was run in each experiment, which represents adifferent donor.

TABLE 3 CD86 CD40 Compound (5 Exp) (4 Exp.) T cell proliferation GM-CSF1.9 2.5 13.3 CpG 3.9 3.5 19.7 CpG + GM-CSF 7.0 8.5 25.6U.S. Pat. No. 6,239,116 B1

TABLE 1 Olionucleotide Stimulation of Mouse B Cells Stimulation Index¹ODN Sequence (5′ to 3′)† ³H Uridine IgM Production 1 (SEQ ID NO: 1)GCTAGACGTTAGCGT 6.1 ± 0.8 17.9 ± 3.6  1a (SEQ. ID NO: 42)......T........ 1.2 ± 0.2 1.7 ± 0.5 1b (SEQ ID NO: 3) ......Z........1.2 ± 0.1 1.8 ± 0.0 1c (SEQ ID NO: 4) ............Z.. 10.3 ± 4.4 9.5 ± 1.8 1d (SEQ ID NO: 5) ..AT......GAGC. 13.0 ± 2.3  18.3 ± 7.5 2 (SEQ ID NO: 6) ATGGAAGGTCCAGCGTTCTC 2.9 ± 0.2 13.6 ± 2.0 2a (SEQ ID NO: 7) ..C..CTC..G......... 7.7 ± 0.8 24.2 ± 3.2 2b (SEQ ID NO: 8) ..Z..CTC.ZG..Z...... 1.6 ± 0.5 2.8 ± 2.22c (SEQ ID NO: 9) ..Z..CTC..G......... 3.1 ± 0.6 7.3 ± 1.42d (SEQ ID NO: 10) ..C..CTC..G......Z.. 7.4 ± 1.4 27.7 ± 5.4 2e (SEQ ID NO: 740) ............A....... 5.6 ± 2.0 ND 3D (SEQ ID NO: 12)GAGAACGCTGGACCTTCCAT 4.9 ± 0.5 19.9 ± 3.6  3Da (SEQ ID NO: 749)..........C......... 6.6 ± 1.5 33.9 ± 6.8  3Db (SEQ ID NO: 14).........C.......G.. 10.1 ± 2.8  25.4 ± 0.8  3Dc (SEQ ID NO: 15)...C.A.............. 1.0 ± 0.1 1.2 ± 0.5 3Dd (SEQ ID NO: 17).....Z.............. 1.2 ± 0.2 1.0 ± 0.4 3De (SEQ ID NO: 12).............Z...... 4.4 ± 1.2 18.8 ± 4.4  3Df (SEQ ID NO: 18).......A............ 1.6 ± 0.1 7.7 ± 0.4 3Dg (SEQ ID NO: 19).........CC.G.ACTG.. 6.1 ± 1.5 18.6 ± 1.5  3M (SEQ ID NO: 22)TCCATGTCGGTCCTGATGCT 4.1 ± 0.2 23.2 ± 4.9  3Ma (SEQ ID NO: 21)......CT............ 0.9 ± 0.1 1.8 ± 0.5 3Mb (SEQ ID NO: 22).......Z............ 1.3 ± 0.3 1.5 ± 0.6 3Mc (SEQ ID NO: 23)...........Z........ 5.4 ± 1.5 8.5 ± 2.6 3Md (SEQ ID NO: 24)......A..T.......... 17.2 ± 9.4  ND 3Me (SEQ ID NO: 741)...............C..A. 3.6 ± 0.2 14.2 ± 5.2  4 (SEQ ID NO: 26) TCAACGTT6.1 ± 1.4 19.2 ± 5.2  4a (SEQ ID NO: 27) ....GC.. 1.1 ± 0.2 1.5 ± 1.14b (SEQ ID NO: 28) ...GCGC. 4.5 ± 0.2 9.6 ± 3.4 4c (SEQ ID NO: 2306)...TCGA. 2.7 ± 1.0 ND 4d (SEQ ID NO: 30) ..TT..AA 1.3 ± 0.2 ND4e (Residue 2-8 of -....... 1.3 ± 0.2 1.1 ± 0.5 SEQ ID NO: 26)(SEQ ID NO: 31) 4f (SEQ ID NO: 32) C....... 3.9 ± 1.4 ND4g (Residue 11-18 of --......CT 1.4 ± 0.3 ND SEQ ID NO: 740)(SEQ ID NO: 2307) 4h (SEQ ID NO: 34) .......C 1.2 ± 0.2 ND LPS 7.8 ± 2.54.8 ± 1.0 ¹Stimulation indexes are the means and std. dev. derived fromat least 3 separate experiments, and are compared to wells cultured withno added ODN. ND = not done. CpG dinuclectides are underlined. Dotsindicate identity; dashes indicate deletions. Z indicates S methylcytosine.

TABLE 2Identification of the optimal CpG motif for Murine IL-6 production andB cell activation IL-6 (pg/ml)^(a) ODN SEQUENCE (5′-3′) CH12.LXSPLENIC B CELL SI^(b) IgM (ng/ml)^(c) 512 (SEQ ID NO: 22)TCCATGTCGTCCTGATGCT 1300 ± 106 627 ± 43 5.8 ± 0.3  7315 ± 1324 1637(SEQ ID NO: 43) ......C............. 136 ± 27 46 ± 6 1.7 ± 0.2 770 ± 721615 (SEQ ID NO: 44) ......G............. 1201 ± 155  850 ± 2023.7 ± 0.3 3212 ± 617 1614 (SEQ ID NO: 45) ......A.............1533 ± 321 1812 ± 103 10.8 ± 0.6  7558 ± 414 1636 (SEQ ID NO: 46).........A.......... 1181 ± 76   947 ± 132 5.4 ± 0.4 3983 ± 485 1634(SEQ ID NO: 47) .........C.......... 1049 ± 223 1671 ± 175 9.2 ± 0.96256 ± 261 1619 (SEQ ID NO: 48) .........T.......... 1555 ± 3042908 ± 129 12.5 ± 1.0  8243 ± 698 1618 (SEQ ID NO: 24)......A..T.......... 2109 ± 291 2596 ± 166 12.9 ± 0.7  10425 ± 674  1639(SEQ ID NO: 49) .....AA...T.......... 1827 ± 83  2012 ± 132 11.5 ± 0.4 9489 ± 103 1707 (SEQ ID NO: 50) ......A..TC......... ND 1147 ± 1754.0 ± 0.2 3534 ± 217 1708 (SEQ ID NO: 51) .....CA..TG......... ND 59 ± 31.5 ± 0.1  466 ± 109 Dots indicate identity; CpG dinucleotides areunderlined; ND = not done ^(a)The experiment was done at least threetimes with similar results. The level of IL-6 of unstimulated controlcultures of both CH12.LX and splenic B cells was ≤10 pg/ml. The IgMlevel of unstimulated culture was 547 ± 82 ng/ml. CpG dinucleotides areunderlined and dots indicate identity. ^(b)[³H] Uridine uptake wasindicated as a fold increase (SI: stimulation index) from unstimulatedcontrol (2322.67 ± 213.68 cpm). Cells were stimulated with 20 μM ofvarious CpG O-ODN. Data present the mean ± SD of triplicates^(c)Measured by ELISA.

TABLE 3 Induction of Murine IL-6 secretion by CpGmotifs in bacterial DNA or oligonucleotides. Treatment IL-6 (pg/ml)calf thymus DNA ≤10 calf thymus DNA + DNase ≤10 E. coli DNA1169.5 ± 94.1 E. coli DNA + DNase ≤10 CpG methylated E. coli DNA ≤10 LPS 280.1 ± 17.1 Media (no DNA) ≤10 ODN 5a SEQ. ID. ATGGACTCTCCAGCGTTCTC1096.4 ± 372.0 No: 35 5b SEQ. ID. .....AGG....A....... 1124.5 ± 126.2No: 740 5c SEQ. ID. ..C.......G......... 1783.0 ± 189.5 No: 7 5dSEQ. ID. .... AGG..C..T...... ≤10 No: 742 5e SEQ. ID...C.......G...Z......  851.1 ± 114.4 No: 2308 5f SEQ. ID...Z......ZG..Z...... ≤10 No: 9 5g SEQ. ID. ..C.......G......Z..1862.3 ± 87.26 No: 10 T cell depleted spleen cells from DBA/2 mice werestimulated with phosphodiester modified oligonucleotides (O-ODN) (20μM), calf thymus DNA (50 μg/ml)or E. Coli DNA (50 μg/ml) with ot withoutenzyme treatment, or LPS (10 μg/ml) for 24 hr. Data represent the mean(pg/ml) ± SD of triplicates. CpG dinucleotides are underlined and dotsindicate identity. Z indicates 5-methylcytosine.

TABLE 4 Secretion of Murine IL-6 induced by CpG DNA stimulation in vivo.Stimulant IL-6 (pg/ml) PBS <50 E. coli DNA 13858 ± 3143 Calf Thymus DNA<50 CpG S-ODN 20715 ± 606  non-CpG S-ODN <50 Mice (2 mice/group) werei.v. injected with 100 μl of PBS, 200 μg of E. coli DNA or calf. thymusDNA, or 500 μg of CpG S-ODN or non-CpG control S-ODN. Mice were bled 2hr after injection and 1:10 dilution of each serum was analyzed by IL-6ELISA. Sensitivity limit of IL-6 ELISA was 5 pg/ml. Sequences of the CpGS-ODN is 5′GCATGACGT-TGAGCT3′ (SEQ. ID. No: 6) and of thenon-stimulatory S-ODN is 5′GCTAGATGTTAGCGT3′ (SEQ. ID. No: 49). Notethat although there is a CpG in sequence 48, it is too close to the3′ end to effect stimulation, as explained herein. Data represent mean± SD of duplicates. The experiment was done at least twice with similarresults.

TABLE 5 Induction of human PBMC cytokine secrtetion by CpG oligos ODNSequence (5′-3′) IL-6₁ TNF-_(α1) IFN-_(γ1) GM-CSF IL-12  512TCCATGTCGGTCCTGATGCT 500 140 15.6 70 250 SEQ ID NO: 22 1637......C............. 550 16 7.8 15.6 16 SEQ ID NO: 43 1615......G............. 600 145 7.8 45 145 SEQ ID NO: 44 1614......A............. 550 31 0 50 31 SEQ ID NO: 45 1636.........A.......... 325 250 35 40 250 SEQ ID NO: 46 1634.........C.......... 300 400 40 85 400 SEQ ID NO: 47 1619.........T.......... 275 450 200 80 450 SEQ ID NO: 48 1618......A..T.......... 300 60 15.6 15.6 62 SEQ ID NO: 24 1639.....AA..T.......... 625 220 15.6 40 220 SEQ ID NO: 49 1707......A..TC......... 300 70 17 0 70 SEQ ID NO: 50 1708.....CA..TG......... 270 10 17 ND 10 SEQ ID NO: 51 dots indicateidentity; CpG dinucleotides are underlined, measured by ELISA usingQuantikine kits from R&D Systems (pg/ml) Cells were caltured in 10%autologous serum with the indicated oligodeoxynucleotides (12 μg/ml) for4 hr in the case of TNF-α or 24 hr for the other cytokines beforesupernatant harvest and assay. Data are presented as the level ofcytokine above that in wells with no added oligodeoxynucleotide.

TABLE 6 CpG DNA induces cytokine secretion by human PBMC TNF- IL-6 IFN-γRANTES DNA α (pg/ml)¹ (pg/ml) (pg/ml) (pg/ml) EC DNA (50 μg/ml) 90012,000 700 1560 EC DNA (5 μg/ml) 850 11,000 400 750 EC DNA (0.5 μ/ml)500 ND 200 0 EC DNA (0.05 μg/ml) 62.5 10,000 15.6 0 EC DNA (50 μg/ml) +0 ND ND ND L-LME² EC DNA (10 μg/ml) Methyl.³ 0 5 ND ND CT DNA (50 μg/ml)0 600 0 0 ¹Levels of all cytokines were determined by ELISA usingQuantikine kits from R&D Systems as described in the previous table.Results are representative using PBMC from different donors. ²Cells werepretreated for 15 min. with L-leucyl-L-leucine methyl ester (M-LME) todetermine whether the cytokine production under these conditions wasfrom monocytes (or other L-LME-sensitive cells). ³EC DNA was methylatedusing 2 U/μg DNA of CpG methylase (New England Biolabs) according to themanufacturer's directions, and methylation confirmed by digestion withHpa-II and Msp-I. As a negative control, samples were includedcontaining twice the maximal amount of LPS contained in the highestconcentration of EC DNA which failed to induce detectable cytokineproduction under these experimental conditions. ND = not done

TABLE 7 CpG DNA induces cytokine expression in purified humanmacrophages IL-6 (pg/ml) GM-CSF (pg/ml) TNF-α(pg/ml) Cells alone 0 0 0CT DNA (50 μg/ml) 0 0 0 EC DNA (50 μg/ml) 2000 15.6 1000

TABLE 8 Induction Of NK Activity By CpG Oligodeoxynucleotides (ODN) %YAC-1 Specific Lysis* % 2C11 Specific Lysis Effector:TargetEffector:Target ODN 50:1 100:1 50:1 100:1 None −1.1 −1.4 15.3 16.6 116.1 24.5 38.7 47.2 3Dd 17.1 27.0 37.0 40.0 non-CpG ODN −1.6 −1.7 14.815.4

TABLE 9Induction of NK Activity by DNA Containing CpG Motifs but not by Non-CpG DNA LU/10⁶ DNA or Cytokine Added Mouse Cells Human Cells Expt. 1None 0.00 0.00 IL-2 16.68 15.82 E. Coli, DNA 7.23 5.05 Calf thymus DNA0.00 0.00 Expt. 2 None 0.00 3.281585 ggGGTCAACGTTGACgggg (SEQ ID No. 52) 7.38 17.981629 -------gtc----- (SEQ ID No. 750) 0.00 4.4 Expt. 3 None 0.001613 GCTAGACGTTAGTGT (SEQ ID No. 54) 5.221769 -------Z----- (SEQ ID No. 745) 0.02 ND1619 TCCATGTCGTTCCTGATGCT (SEQ ID No: 48) 3.351765 -------Z--------- (SEQ ID No. 56) 0.11 CpG dinuoleotides in ODNsequences are indicated by underlying; Z indicates methylcytosine. Lowercase letters indicate nuclease resistant phosphorothioate modifiedinternucleotide linkages which, in titration experiments, were more than20 times as potent as non-modified ODN, depending on the flanking bases.Poly G ends (g) were used in some ODN, because they significantlyincrease the level of ODN uptake.

TABLE 10 ODN induction of NK Lytic Activity (LU) ODN cells LU aloneSequence (5′-3′) 0.01 1754 ACCATGGACGATCTGTTTCCCCTC 0.02 SEQ ID NO: 581758 TCTCCCAGCGTGCGCCAT 0.05 SEQ ID NO: 59 1761 TACCGCGTGCGACCCTCT 0.05SEQ ID NO: 60 1776 ACCATGGACGAACTGTTTCCCCTC 0.03 SEQ ID NO: 61 1777ACCATGGACGAGCTGTTTCCCCTC 0.05 SEQ ID NO: 62 1778ACCATGGACGTACTGTTTCCCCTC 0.01 SEQ ID NO: 63 1779ACCATGGACGTACTGTTTCCCCTC 0.02 SEQ ID NO: 64 1780ACCATGGACGGTCTGTTTCCCCTC 0.29 SEQ ID NO: 65 1781ACCATGGACGTTCTGTTTCCCCTC 0.38 SEQ ID NO: 66 1823 GCATGACGTTGAGCT 0.08SEQ ID NO: 41 1824 CACGTTGAGGGGCAT 0.01 SEQ ID NO: 67 1825CTGCTGAGACTGGAG 0.01 SEQ ID NO: 68 1828 TCAGCGTGCGCC 0.01 SEQ ID NO: 691829 ATGACGTTCCTGACGTT 0.42 SEQ ID NO: 70 1830² RANDOM SEQUENCE 0.251834 TCTCCCAGCGGGCGCAT 0.00 SEQ ID NO: 71 1836 TCTCCCAGCGCGCGCCAT 0.46SEQ ID NO: 72 1840 TCCATGTCGTTCCTGTCGTT 2.70 SEQ ID NO: 73 1841TCCATAGCGTTCCTAGCGTT 1.45 SEQ ID NO: 74 1842 TCGTCGCTGTCTCCGCTTCTT 0.06SEQ ID NO: 75 1851 TCCTGACGTTCCTGACGTT 2.32 SEQ ID NO: 76 ¹Lytic unite(LU) were measured as described (8). Briefly, PBMC were collected fromnormal donors and spun over Ficoll, then cultured with or without theindicated ODN (which were added to cultures at 6 μg/ml) for 24 hr. Thentheir ability to lyse ⁵¹Cr-labeled K562 cells was determined. Theresults shown are typical of those obtained with several differentnormal human donors. ²This oligo mixture contained a random selection ofall 4 bases at each position.

TABLE 11 Induction of NK LU by Phoshorothioate CpG ODN with Good MotifsODN¹ cells expt. 1 expt. 2 expt. 3 alone sequence (5′-3′) SEQ ID NO:0.00 1.26 0.46 1840 TCCATGTCGTTCCTGTCGTT 73 2.33 ND ND 1960TCCTGTCGTTCCTGTCGTT 77 ND 0.48 8.99 1961 TCCATGTCGTTTTTGTCGTT 78 4.031.23 5.08 1962 TCCTGTCGTTCCTTGTCGTT 79 ND 1.60 5.74 1963TCCTTGTCGTTCCTGTCGTT 80 3.42 ND ND 1965 TCCTGTCGTTTTTTGTCGTT 81 0.460.42 3.48 1966 TCGTCGCTGTCTCCGCTTCTT 75 2.62 ND ND 1967TCGTCGCTGTCTGCCCTTCTT 82 5.82 1.64 8.32 1968 TCGTCGCTGTTGTCGTTTCTT 833.77 5.26 5.12 1979² TCCATGTZGTTCCTGTZGTT 84 1.32 ND ND 1982TCCAGGACTTCTCTCAAGTT 751 0.05 ND 0.98 1990 TCCATGCGTGCGTGCGTTTT 86 2.10ND ND 1991 TCCATGCGTTGCGTTGCGTT 87 0.89 ND ND 2002 TCCACGACGTTTTCGACGTT88 4.02 1.31 9.79 2005 TCGTCGTTGTCGTTGTCGTT 89 ND 4.22 12.73  2006TCGTCGTTTTGTCGTTTTGTCGT 752 ND 6.17 12.82  2007 TCGTCGTTGTCGTTTTGTCGTT91 ND 2.68 9.65 2008 GCGTGCGTTGTCGTTGTCGTT 92 ND 1.37 8.15 2010GCGGCGGGCGGCGCGCGCCC 93 ND 0.01 0.05 2012 TGTCGTTTGTCGTTTGTCGTT 94 ND2.02 11.61  2013 TGTCGTTGTCGTTGTCGTTGTCGTT 95 ND 0.56 5.22 2014TGTCGTTGTCGTTGTCGTT 96 ND 5.74 10.89  2015 TCGTCGTCGTCGTT 97 ND 4.5310.13  2016 TGTCGTTGTCGTT 98 ND 6.54 8.06 ¹PBMC essentially as describedherein. Results are representative of 6 separate experiments; eachexperiment represents a different donor. ²This is the methylated versionof ODN 1840 (SEQ ID NO: 83); Z = 5-methyl cytosine at residues 8 and 17;LU is lytic units; ND = not done; CpG dinucleotides are underlined forclarity

TABLE 12Induction of human B cell proliferation by Phosphorothicate CpG ODNStimulation Index^(1L) ODN sequence (5′ 3′) SEQ ID NO: expt. 1 expt. 2expt. 3 expt. 4 expt. 5 expt. 6 1840 TCCATGTCGTTCCTGTCGTT 73 4 ND ND NDND 34 1841 TCCATAGCGTTCCTAGCGTT 74 3 ND ND ND ND ND 1960TCCTGTCGTTCCTGTCGTT 77 ND 2.0 2.0 3.6 ND ND 1961 TCCATGTCGTTTTTGTCGTT 782 3.9 1.9 3.7 ND 37 1962 TCCTGTCGTTCCTTGTCGTT 79 ND 3.9 1.9 3.9 5.4 351963 TCCTTGTCGTTCCTGTCGTT 80 3 ND ND ND ND ND 1965 TCCTGTCGTTTTTTGTCGTT81 4 3.7 2.4 4.7 6.0 43 1967 TCGTCGTGTCTGCCCTTCTT 82 ND 4.4 2.0 4.5 5.038 1968 TCGTCGCTGTGTCGTTTCTT 83 ND 4.0 2.0 4.9 8.7 38 1982TCCAGGACTTCTCTCAGGTT 85 3 1.8 1.3 3.1 3.2 12 2002 TCCACGACGTTTTCGACGTT88 ND 2.7 1.4 4.4 ND 14 2005 TCGTCGTTGTCGTTGTCGTT 89 5 3.2 1.2 3.0 7.937 2006 TCGTCGTTTTGTCGTTTTGTCGTT 90 4 4.5 2.2 5.8 8.3 40 2007TCGTCGTTGTCGTTTTGTCGTT 91 3 4.0 4.2 4.1 ND 22 2008 GCGTGCGTTGTCGTTGTCGTT92 ND 3.0 2.4 1.6 ND 12 2010 GCGGCGGGCGGCGCGCGCCC 93 ND 1.6 1.9 3.2 NDND 2012 TGTCGTTTGTCGTTTGTCGTT 94 2 2.8 0 3.2 ND 33 2013TGTCGTTGTCGTTGTCGTTGTCGTT 95 3 2.3 3.1 2.8 ND  7 2014TGTCGTTGTCGTTGTCGTT 96 3 2.5 4.0 3.2 6.7 14 2015 TCGTCGTCGTCGTT 97 5 1.82.6 4.5 9.4  1 2016 TGTCGTTGTCGTT 98 ND 1.1 1.7 2.7 7.3  1 ¹Cells= human spleen cells stored at −70° C. after surgical harvest or PBNCcollected from normal donors and spun over Ficoll. Cells were culturedin 96 well U-bottom microtiter lates with or without the indicated ODN(which were added to cultures at 6 μml). N = 12 experiments. Cells werecultured for 4-7 days, pulsed with 1 μCi of ³H thymidine for 18 hrbefore harvest and scintillation counting. Stimulation index = the ratioof cpm in wells without ODN to that in wells that had been stimulatedthroughout the culture period with the indicated ODN (there were nofurther additions of ODN after the culture were set up). ND = not done

TABLE 13 Induction of human IL-12 secretion by Phophorothicate CpG ODNSEQ IL-12 (pg/ml) ID expt. expt. ODN¹ sequence (5′-3′) no 1 2 cells 0 0alone 1962 TCCTGTCGTTCCTTGTCGTT 79 19 0 1965 TCCTGTCGTTTTTTGTCGTT 81 360 1967 TCGTCGCTGTCTGCCCTTCTT 82 41 0 1968 TCGTCGCTGTTGTCGTTTCTT 83 24 02005 TCGTCGTTGTCGTTGTCGTT 89 25 0 2006 TCGTCGTTTTGTCGTTTTGTCGTT 90 29 152014 TGTCGTTGTCGTTGTCGTT 96 28 0 2015 TCGTCGTCGTCGTT 97 14 0 2016TGTCGTTGTCGTT 98 3 0 ¹PBMC were collected from normal donors and spunover Ficoll, then cultured at 10⁶ cells/well in 96 well microtiterplates with or without the indicated ODN which were added to cultures at6 μg/ml. Supernatants were collected at 24 hr and tested for IL-12levels by ELISA as described in methods. A standard curve was run ineach experiment, which represents a different donor.

TABLE 14 Different CpG motifs stimulate optimalmurine B cell and NK activation B cell NK ODN Sequence activation¹activation² 1668 TCCATGACGTTCCTGATGCT 42,849 2.52 (SEQ. ID. NO: 24) 1758TCTCCCAGCGTGCGCCAT 1,747 6.66 (SEQ. ID. NO: 59) NONE 367 0.00 CpGdinucleotides are underlined; oligonucleotides were synthesized withphosphorothioate modified backbones to improve their nucleaseresistance. ¹Measured by ³H thymidine incorporation after 48 hr culturewith oligodeoxynucleotides at a 200 nM concentration as described inExample 1. ²Measured in lytic units.

TABLE 15Specific blockade of CpG-induced TNF-α and IL-12 expression by inhibitorsof endosomal acidification or NFκB activation Inhibitors: Glio- Bisglio-Bafilomycin Chloroquine Monensin NAC TPCK toxin toxin Medium (250 nM)(2.5 μg/ml) (10 μM) (50 mM) (50 μM) (0.1 μg/ml) (0.1 μg/ml) activatorsTNF-α IL-12 TNF-α IL-12 TNF-α IL-12 TNF-α IL-12 TNF-α TNF-α TNF-α TNF-αMedium 37 147 46 102 27 20 22 73 10 24 17 41 CpG 455 17,114 71 116 28 649 777 54 23 31 441 ODN LPS 901 22,485 1370 4051 1025 12418 491 4796 41746 178 1120 TABLE 15 legend IL-12 and TNF-α assays: The murine monocytecell line 3774 (1 × 10⁵ cells/ml for IL 12 or 1 × 10⁶ cells/ml forTNF-α), were cultured with or without the indicated inhibitors at theconcentrations shown for 2 hr and then stimulated with the CpGoligodeoxynucleotide (ODN) 1826 (TCCATGACGTTCCTGACGTT SEQ ID NO: 100) at2 μm or LPS (10 μg/ml) for 4 hr (TNF-α or 24 hr (IL-12) at which timethe supernatant was harvested. ELISA for IL-12 or TNF-α (pg/ml) wasperformed on the supernatants essentially as described (A.K. Krieg,A.-K. Yi, S. Matson, T.J. Waldschmidt, G.A. Bishop, R. Teasdale, G.Koretzy and D. Klinman, Nature 374, 546 (1995); Yi A.-K., D.M. Klinman,T.L. Martin, S. Matson and A.M. Krieg. J Immunol., 157, 5394-5402(1996); Krieg, A.M, J Lab. Clin. Med., 128, 128-133 (1996). Cellscultured with ODN that lacked CpG motifs did not induce cytokinesecretion. Similar specific inhibition of CpG responses was seen withIL-6 assays, and in experiments using primary spleen cells or the B celllines CH12.LX and WEHI-231.2.5 μg/ml of chloroquine is equivalent to <5μM. Other inhibitors of NF-κB activation including PDTC and calpaininhibitors I and II gave similar results to the inhibitors shown. Theresults shown are representative of those obtained in ten differentexperiments.U.S. Pat. No. 6,339,068 B1

TABLE 3 Plasmids containing immunostimulatory CpG motifsSpecies specificity and No. CpG, ODN Equivalence of CpG-S PlasmidBackbone Motifs Insert pMCG-16 pMAS 16 mouse-specific CpG motif pMCG-50pMAS 50 #1826¹ pMCG- pMAS 300 100 pMCG- pMAS 200 200 pHCG-30 pMAS 30human-specific CpG motif- pHCG-50 pMAS 50 no ODN equivalent² pHCG- pMAS100 100 pHCG- pMAS 200 200 pHIS-40 pMAS 40 human-specific CpG motifpHIS-64 pMAS 64 #2006³ pHIS- pMAS 128 128 pHIS- pMAS 192 192 ¹sequenceof 1826 is TCCATGACGTTCCTGACGTT (SEQ ID NO. 100) ²sequence used as asource of CpG motifs is GACTT CGTGTCGTTCTTCTGTCGTCTTTAGCGCTTCTCCTGCGTGCGTCCCTTG(SEQ ID NO: 753) ³sequence of 2006 isTCGTCGTTTTGTCGTTTTGTCGTT(SEQ ID NO: 90)

TABLE 4 Plasmids encoding hepatitis B surface antigen(derived from ayw or adw subtypes of HBV) Plasmid Backbone Insert pUK-SpUK21-A2 HBV-S (ayw) pUKAX-S pUK21-AX* HBV-S (ayw) pMAS-S pMASHBV-S (ayw) pMCG16-S pMCG-16 HBV-S (ayw) pMCG50-S pMCG-50 HBV-S (ayw)pMCG100-S pMCG-100 HBV-S (ayw) pMCG200-S pMCG-200 HBV-S (ayw) pHCG30-SpHCG-30 HBV-S (ayw) pHCG50-S pHCG-50 HBV-S (ayw) pHCG100-S pHCG-100HBV-S (ayw) pHCG200-S pHCG-200 HBV-S (ayw) pHIS40-S(ad) pHIS-40HBV-S(adw2) pHIS64-S(ad) pHIS-64 HBV-S(adw2) pHIS128-S(ad) pHIS-128HBV-S(adw2) pHIS192-S(ad) pHIS-192 HBV-S(adw2) *pUK21-AX was created bydeleting f1 origin from pUK21-A

TABLE 6 ODN used with plasmid DNA ODN Backbone code number SequenceS-ODN 1826 (SEQ ID NO: 100) TCCATGACGTTCCTGACGTT 1628 (SEQ ID NO: 754)GGGGTCAACGTTGAGGGGGG 1911 (SEQ ID NO: 755) TCCAGGACTTTCCTCAGGTT 1982(SEQ ID NO: 85) TCCAGGACTTCTCTCAGGTT 2017 (SEQ ID NO: 756)CCCCCCCCCCCCCCCCCCCC O-ODN 2061 (SEQ ID NO: 100) TCCATGACGTTCCTGACGTT2001 (SEQ ID NO: 757) GGCGGCGGCGGCGGCGGCGG SOS-ODN 1980 (SEQ ID NO: 100)TCCATGACGTTCCTGACGTT 1585 (SEQ ID NO: 758) GGGTCAACGTTGAGGGGGG 1844(SEQ ID NO: 759) TCTCCCAGCGTGCGCCATAT 1972 (SEQ ID NO: 760)GGGGTCTGTGCTTTTGGGGGG 2042 (SEQ ID NO: 761) TCAGGGGTGGGGGGAACCTT 1981(SEQ ID NO: 762) GGGGTTGACGTTTTGGGGGG 2018 (SEQ ID NO: 763)TCTAGCGTTTTTAGCGTTCC 2021 (SEQ ID NO: 89) TCGTCGTTGTCGTTGTCGTT 2022(SEQ ID NO: 90) TCGTCGTTTTGTCGTTTTGTCGTT 2023 (SEQ ID NO: 91)TCGTCGTTGTCGTTTTGTCGTT

TABLE 10 Inhibitory CpG motifs can block B cellproliferation induced by a stimulatory CpG motif Oligonucleotide addedmedium 194 1968 (TCCATGACGTTCCTGATGCT 34,669 (SEQ ID NO: 24) 24,452 1668+1735 (GCGTTTTTTTTTGCG) 601 (SEQ ID NO: 764) 1720 (TCCATGAGCTTCCTGATGCT)1109 (SEQ ID NO: 765) 1720 +1735 Inhibitory effects of “bad” CpGmotifs on the “good” CpG Oligo 1619 I: −12 in Oligonucleotide addedpg/ml medium 0 1619 alone 6 1619 1949 (TCCATGTCGTTCCTGATGCG 16(SEQ ID NO: 766)) 1619 1952 (TCCATGTCGTTCCGCGCGCG 0 (SEQ ID NO: 767))1619 1953 (TCCATGTCGTTCCTGCCGCT 0 (SEQ ID NO: 768)) 16191953 (GCGGCGGGCGGCGCGCGCCC 0 (SEQ ID NO: 93)) ¹Dots 1720in the sequenceof ODN 1952 and 1953 indicate identity to ODN 1619; CpG dinucleotidesare underlined for clarity. ODN without CpG-N or CpG-S motifs had littleor no effect on cytokine production. The data shown are representativeof 4 experiments. ²All cytokines are given in pg/ml; measured by ELISAon supernatants from DBA/2 spleen cells cultured in 96 well plates at 2× 10⁷ cells/ml for 24 hr with the indicated ODN at 30 μg/ml. Std. dev.of the triplicate wells was <7%. None of the ODN induced significantamounts of IL-5.

TABLE 13 Identification of neutralizing CpG motifswhich reduce the induction of cytokinesecretion by a CpG-S motif in the same ODN (cis-neutralization)ODN-induced cytokine expression² ODN sequence 5′-3′¹ IL-6² IL-12 IFN-γNone <5 206 898 1619 TCCATGTCGTTCCTGATGCT 1405 3130 4628 (SEQ ID NO: 48)1952 .............GCGCGCG 559 1615 2135 (SEQ ID NO: 767) 1953...............CC... (SEQ ID NO: 768) ¹Dots in the sequence of ODN 1952and 1953 indicate identity to ODN 1619; CpG dinucleotides are underlinedfor clarity. ODN without CpG-N or CpG-S motifs had little or no effecton cytokine production. The data shown are representative of 4experiments. ²All cytokines are given in pg/ml; measured by ELISA onsupernatants from DBA/2 spleen cells cultured in 96 well plates at 2× 10⁷ cells/ml for 24 hr with the indicated ODN at 30 μg/ml. Std. dev.of the triplicate wells was <7%. None of the ODN induced significantamounts of IL-5.

TABLE 14 Inhibition of CpG-induced cytokinesecretion by ODN containing CpG-N motifs CpG-S- induced IL-12 IL-12 ODNsequence 5′-3′ secretion¹  secretion² none 268 5453 1895GCGCGCGCGCGCGCGCGCGC 123 2719 (SEQ ID NO: 769) 1896 CCGGCCGGCCGGCCGGCCGG292 2740 (SEQ ID NO: 770) 1955 GCGGCGGGCGGCGCGCGCCC 270 2539(SEQ ID NO: 93) 2037 TCCATGCCGTTCCTGCCGTT 423 2847 (SEQ ID NO: 771)¹BALB/c spleen cells were cultured in 96 well plates at 2 × 10⁷ cells/mlwith the indicated ODN for 24 hr and then the supernatants were assayedfor IL-12 by ELISA (pg/ml). ²Cells were set up the same as in ¹ exceptthat IL-12 secretion was induced by the addition of the CpG ODN 1619(TCCATGTCGTTCCTGATGCT) (SEQ ID NO: 48) at 30 μg/ml. The data shown arerepresentative of 5 experiments.

APPENDIX (C)

Exemplary Human miRNA Sequences >hsa-let-7a-1 MI0000060 (SEQ ID NO: 772)UGGGAUGAGGUAGUAGGUUGUAUAGUUUUAGGGUCACACCCACCACUGGGAGAUAACUAUACAAUCUACUGUCUUUCCUA >hsa-let-7a-2 MI0000061 (SEQ ID NO: 773)AGGUUGAGGUAGUAGGUUGUAUAGUUUAGAAUUACAUCAAGGGAGAUAACUGUACAGCCUCCUAGCUUUCCU >hsa-let-7a-3 MI0000062 (SEQ ID NO: 774)GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUCUGCCCUGCUAUGGGAUAACUAUACAAUCUACUGUCUUUCCU >hsa-let-7b MI0000063 (SEQ ID NO: 775)CGGGGUGAGGUAGUAGGUUGUGUGGUUUCAGGGCAGUGAUGUUGCCCCUCGGAAGAUAACUAUACAACCUACUGCCUUCCCUG >hsa-let-7c MI0000064 (SEQ ID NO: 776)GCAUCCGGGUUGAGGUAGUAGGUUGUAUGGUUUAGAGUUACACCCUGGGAGUUAACUGUACAACCUUCUAGCUUUCCUUGGAGC >hsa-let-7d MI0000065 (SEQ ID NO: 777)CCUAGGAAGAGGUAGUAGGUUGCAUAGUUUUAGGGCAGGGAUUUUGCCCACAAGGAGGUAACUAUACGACCUGCUGCCUUUCUUAGG >hsa-let-7e MI0000066 (SEQ ID NO: 778)CCCGGGCUGAGGUAGGAGGUUGUAUAGUUGAGGAGGACACCCAAGGAGAUCACUAUACGGCCUCCUAGCUUUCCCCAGG >hsa-let-7f-1 MI0000067 (SEQ ID NO: 779)UCAGAGUGAGGUAGUAGAUUGUAUAGUUGUGGGGUAGUGAUUUUACCCUGUUCAGGAGAUAACUAUACAAUCUAUUGCCUUCCCUGA >hsa-let-7f-2 MI0000068 (SEQ ID NO: 780)UGUGGGAUGAGGUAGUAGAUUGUAUAGUUUUAGGGUCAUACCCCAUCUUGGAGAUAACUAUACAGUCUACUGUCUUUCCCACG >hsa-let-7g MI0000433 (SEQ ID NO: 781)AGGCUGAGGUAGUAGUUUGUACAGUUUGAGGGUCUAUGAUACCACCCGGUACAGGAGAUAACUGUACAGGCCACUGCCUUGCCA >hsa-let-7i MI0000434 (SEQ ID NO: 782)CUGGCUGAGGUAGUAGUUUGUGCUGUUGGUCGGGUUGUGACAUUGCCCGCUGUGGAGAUAACUGCGCAAGCUACUGCCUUGCUA >hsa-mir-1-1 MI0000651 (SEQ ID NO: 783)UGGGAAACAUACUUCUUUAUAUGCCCAUAUGGACCUGCUAAGCUAUGGAAUGUAAAGAAGUAUGUAUCUCA >hsa-mir-1-2 MI0000437 (SEQ ID NO: 784)ACCUACUCAGAGUACAUACUUCUUUAUGUACCCAUAUGAACAUACAAUGCUAUGGAAUGUAAAGAAGUAUGUAUUUUUGGUAGGC >hsa-mir-7-1 MI0000263 (SEQ ID NO: 785)UUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUAGUGAUUUUGUUGUUUUUAGAUAACUAAAUCGACAACAAAUCACAGUCUGCCAUAUGGCACAGGCCAUGCCUCUACAG >hsa-mir-7-2 MI0000264 (SEQ ID NO: 786)CUGGAUACAGAGUGGACCGGCUGGCCCCAUCUGGAAGACUAGUGAUUUUGUUGUUGUCUUACUGCGCUCAACAACAAAUCCCAGUCUACCUAAUGGUGCCAGCCAUCGCA >hsa-mir-7-3 MI0000265 (SEQ ID NO: 787)AGAUUAGAGUGGCUGUGGUCUAGUGCUGUGUGGAAGACUAGUGAUUUUGUUGUUCUGAUGUACUACGACAACAAGUCACAGCCGGCCUCAUAGCGCAGACUCCCUUCGAC >hsa-mir-9-1 MI0000466 (SEQ ID NO: 788)CGGGGUUGGUUGUUAUCUUUGGUUAUCUAGCUGUAUGAGUGGUGUGGAGUCUUCAUAAAGCUAGAUAACCGAAAGUAAAAAUAACCCCA >hsa-mir-9-2 MI0000467 (SEQ ID NO: 789)GGAAGCGAGUUGUUAUCUUUGGUUAUCUAGCUGUAUGAGUGUAUUGGUCUUCAUAAAGCUAGAUAACCGAAAGUAAAAACUCCUUCA >hsa-mir-9-3 MI0000468 (SEQ ID NO: 790)GGAGGCCCGUUUCUCUCUUUGGUUAUCUAGCUGUAUGAGUGCCACAGAGCCGUCAUAAAGCUAGAUAACCGAAAGUAGAAAUGAUUCUCA >hsa-mir-10a MI0000266 (SEQ ID NO: 791)GAUCUGUCUGUCUUCUGUAUAUACCCUGUAGAUCCGAAUUUGUGUAAGGAAUUUUGUGGUCACAAAUUCGUAUCUAGGGGAAUAUGUAGUUGACAUAAACACUCCGCUCU >hsa-mir-10b MI0000267 (SEQ ID NO: 792)CCAGAGGUUGUAACGUUGUCUAUAUAUACCCUGUAGAACCGAAUUUGUGUGGUAUCCGUAUAGUCACAGAUUCGAUUCUAGGGGAAUAUAUGGUCGAUGCAAAAACUUCA >hsa-mir-15a MI0000069 (SEQ ID NO: 793)CCUUGGAGUAAAGUAGCAGCACAUAAUGGUUUGUGGAUUUUGAAAAGGUGCAGGCCAUAUUGUGCUGCCUCAAAAAUACAAGG >hsa-mir-15b MI0000438 (SEQ ID NO: 794)UUGAGGCCUUAAAGUACUGUAGCAGCACAUCAUGGUUUACAUGCUACAGUCAAGAUGCGAAUCAUUAUUUGCUGCUCUAGAAAUUUAAGGAAAUUCAU >hsa-mir-16-1 MI0000070 (SEQ ID NO: 795)GUCAGCAGUGCCUUAGCAGCACGUAAAUAUUGGCGUUAAGAUUCUAAAAUUAUCUCCAGUAUUAACUGUGCUGCUGAAGUAAGGUUGAC >hsa-mir-16-2 MI0000115 (SEQ ID NO: 796)GUUCCACUCUAGCAGCACGUAAAUAUUGGCGUAGUGAAAUAUAUAUUAAACACCAAUAUUACUGUGCUGCUUUAGUGUGAC >hsa-mir-17 MI0000071 (SEQ ID NO: 797)GUCAGAAUAAUGUCAAAGUGCUUACAGUGCAGGUAGUGAUAUGUGCAUCUACUGCAGUGAAGGCACUUGUAGCAUUAUGGUGAC >hsa-mir-18a MI0000072 (SEQ ID NO: 798)UGUUCUAAGGUGCAUCUAGUGCAGAUAGUGAAGUAGAUUAGCAUCUACUGCCCUAAGUGCUCCUUCUGGCA >hsa-mir-18b MI0001518 (SEQ ID NO: 799)UGUGUUAAGGUGCAUCUAGUGCAGUUAGUGAAGCAGCUUAGAAUCUACUGCCCUAAAUGCCCCUUCUGGCA >hsa-mir-19a MI0000073 (SEQ ID NO: 800)GCAGUCCUCUGUUAGUUUUGCAUAGUUGCACUACAAGAAGAAUGUAGUUGUGCAAAUCUAUGCAAAACUGAUGGUGGCCUGC >hsa-mir-19b-1 MI0000074 (SEQ ID NO: 801)CACUGUUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGCUGUGUGAUAUUCUGCUGUGCAAAUCCAUGCAAAACUGACUGUGGUAGUG >hsa-mir-19b-2 MI0000075 (SEQ ID NO: 802)ACAUUGCUACUUACAAUUAGUUUUGCAGGUUUGCAUUUCAGCGUAUAUAUGUAUAUGUGGCUGUGCAAAUCCAUGCAAAACUGAUUGUGAUAAUGU >hsa-mir-20a MI0000076 (SEQ ID NO: 803)GUAGCACUAAAGUGCUUAUAGUGCAGGUAGUGUUUAGUUAUCUACUGCAUUAUGAGCACUUAAAGUACUGC >hsa-mir-20b MI0001519 (SEQ ID NO: 804)AGUACCAAAGUGCUCAUAGUGCAGGUAGUUUUGGCAUGACUCUACUGUAGUAUGGGCACUUCCAGUACU >hsa-mir-21 MI0000077 (SEQ ID NO: 805)UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA >hsa-mir-22 MI0000078 (SEQ ID NO: 806)GGCUGAGCCGCAGUAGUUCUUCAGUGGCAAGCUUUAUGUCCUGACCCAGCUAAAGCUGCCAGUUGAAGAACUGUUGCCCUCUGCC >hsa-mir-23a MI0000079 (SEQ ID NO: 807)GGCCGGCUGGGGUUCCUGGGGAUGGGAUUUGCUUCCUGUCACAAAUCACAUUGCCAGGGAUUUCCAACCGACC >hsa-mir-23b MI0000439 (SEQ ID NO: 808)CUCAGGUGCUCUGGCUGCUUGGGUUCCUGGCAUGCUGAUUUGUGACUUAAGAUUAAAAUCACAUUGCCAGGGAUUACCACGCAACCACGACCUUGGC >hsa-mir-23c MI0016010 (SEQ ID NO: 809)AGUGACUUUCCAGGUGUCACACAGUGAGUGGCAUAAUCAGAGUACAAUUUGAGUCAUGCCCAUACAUCACAUUGCCAGUGAUUACCCAAGGAAAGUGACG >hsa-mir-24-1 MI0000080 (SEQ ID NO: 811)CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCAUUUUACACACUGGCUCAGUUCAGCAGGAACAGGAG >hsa-mir-24-2 MI0000081 (SEQ ID NO: 812)CUCUGCCUCCCGUGCCUACUGAGCUGAAACACAGUUGGUUUGUGUACACUGGCUCAGUUCAGCAGGAACAGGG >hsa-mir-25 MI0000082 (SEQ ID NO: 813)GGCCAGUGUUGAGAGGCGGAGACUUGGGCAAUUGCUGGACGCUGCCCUGGGCAUUGCACUUGUCUCGGUCUGACAGUGCCGGCC >hsa-mir-26a-1 MI0000083 (SEQ ID NO: 814)GUGGCCUCGUUCAAGUAAUCCAGGAUAGGCUGUGCAGGUCCCAAUGGGCCUAUUCUUGGUUACUUGCACGGGGACGC >hsa-mir-26a-2 MI0000750 (SEQ ID NO: 815)GGCUGUGGCUGGAUUCAAGUAAUCCAGGAUAGGCUGUUUCCAUCUGUGAGGCCUAUUCUUGAUUACUUGUUUCUGGAGGCAGCU >hsa-mir-26b MI0000084 (SEQ ID NO: 816)CCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUUGUGUGCUGUCCAGCCUGUUCUCCAUUACUUGGCUCGGGGACCGG >hsa-mir-27a MI0000085 (SEQ ID NO: 817)CUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAGGGUCCACACCAAGUCGUGUUCACAGUGGCUAAGUUCCGCCCCCCAG >hsa-mir-27b MI0000440 (SEQ ID NO: 817)ACCUCUCUAACAAGGUGCAGAGCUUAGCUGAUUGGUGAACAGUGAUUGGUUUCCGCUUUGUUCACAGUGGCUAAGUUCUGCACCUGAAGAGAAGGUG >hsa-mir-28 MI0000086 (SEQ ID NO: 818)GGUCCUUGCCCUCAAGGAGCUCACAGUCUAUUGAGUUACCUUUCUGACUUUCCCACUAGAUUGUGAGCUCCUGGAGGGCAGGCACU >hsa-mir-29a MI0000087 (SEQ ID NO: 819)AUGACUGAUUUCUUUUGGUGUUCAGAGUCAAUAUAAUUUUCUAGCACCAUCUGAAAUCGGUUAU >hsa-mir-29b-1 MI0000105 (SEQ ID NO: 820)CUUCAGGAAGCUGGUUUCAUAUGGUGGUUUAGAUUUAAAUAGUGAUUGUCUAGCACCAUUUGAAAUCAGUGUUCUUGGGGG >hsa-mir-29b-2 MI0000107 (SEQ ID NO: 821)CUUCUGGAAGCUGGUUUCACAUGGUGGCUUAGAUUUUUCCAUCUUUGUAUCUAGCACCAUUUGAAAUCAGUGUUUUAGGAG >hsa-mir-29c MI0000735 (SEQ ID NO: 822)AUCUCUUACACAGGCUGACCGAUUUCUCCUGGUGUUCAGAGUCUGUUUUUGUCUAGCACCAUUUGAAAUCGGUUAUGAUGUAGGGGGA >hsa-mir-30a MI0000088 (SEQ ID NO: 823)GCGACUGUAAACAUCCUCGACUGGAAGCUGUGAAGCCACAGAUGGGCUUUCAGUCGGAUGUUUGCAGCUGC >hsa-mir-30b MI0000441 (SEQ ID NO: 824)ACCAAGUUUCAGUUCAUGUAAACAUCCUACACUCAGCUGUAAUACAUGGAUUGGCUGGGAGGUGGAUGUUUACUUCAGCUGACUUGGA >hsa-mir-30c-1 MI0000736 (SEQ ID NO: 825)ACCAUGCUGUAGUGUGUGUAAACAUCCUACACUCUCAGCUGUGAGCUCAAGGUGGCUGGGAGAGGGUUGUUUACUCCUUCUGCCAUGGA >hsa-mir-30c-2 MI0000254 (SEQ ID NO: 826)AGAUACUGUAAACAUCCUACACUCUCAGCUGUGGAAAGUAAGAAAGCUGGGAGAAGGCUGUUUACUCUUUCU >hsa-mir-30d MI0000255 (SEQ ID NO: 827)GUUGUUGUAAACAUCCCCGACUGGAAGCUGUAAGACACAGCUAAGCUUUCAGUCAGAUGUUUGCUGCUAC >hsa-mir-30e MI0000749 (SEQ ID NO: 828)GGGCAGUCUUUGCUACUGUAAACAUCCUUGACUGGAAGCUGUAAGGUGUUCAGAGGAGCUUUCAGUCGGAUGUUUACAGCGGCAGGCUGCCA >hsa-mir-31 MI0000089 (SEQ ID NO: 829)GGAGAGGAGGCAAGAUGCUGGCAUAGCUGUUGAACUGGGAACCUGCUAUGCCAACAUAUUGCCAUCUUUCC >hsa-mir-32 MI0000090 (SEQ ID NO: 830)GGAGAUAUUGCACAUUACUAAGUUGCAUGUUGUCACGGCCUCAAUGCAAUUUAGUGUGUGUGAUAUUUUC >hsa-mir-33a MI0000091 (SEQ ID NO: 831)CUGUGGUGCAUUGUAGUUGCAUUGCAUGUUCUGGUGGUACCCAUGCAAUGUUUCCACAGUGCAUCACAG >hsa-mir-33b MI0003646 (SEQ ID NO: 832)GCGGGCGGCCCCGCGGUGCAUUGCUGUUGCAUUGCACGUGUGUGAGGCGGGUGCAGUGCCUCGGCAGUGCAGCCCGGAGCCGGCCCCUGGCACCAC >hsa-mir-34a MI0000268 (SEQ ID NO: 833)GGCCAGCUGUGAGUGUUUCUUUGGCAGUGUCUUAGCUGGUUGUUGUGAGCAAUAGUAAGGAAGCAAUCAGCAAGUAUACUGCCCUAGAAGUGCUGCACGUUGUGGGGCCC >hsa-mir-34b MI0000742 (SEQ ID NO: 834)GUGCUCGGUUUGUAGGCAGUGUCAUUAGCUGAUUGUACUGUGGUGGUUACAAUCACUAACUCCACUGCCAUCAAAACAAGGCAC >hsa-mir-34c MI0000743 (SEQ ID NO: 835)AGUCUAGUUACUAGGCAGUGUAGUUAGCUGAUUGCUAAUAGUACCAAUCACUAACCACACGGCCAGGUAAAAAGAUU >hsa-mir-92a-1 MI0000093 (SEQ ID NO: 836)CUUUCUACACAGGUUGGGAUCGGUUGCAAUGCUGUGUUUCUGUAUGGUAUUGCACUUGUCCCGGCCUGUUGAGUUUGG >hsa-mir-92a-2 MI0000094 (SEQ ID NO: 837)UCAUCCCUGGGUGGGGAUUUGUUGCAUUACUUGUGUUCUAUAUAAAGUAUUGCACUUGUCCCGGCCUGUGGAAGA >hsa-mir-92b MI0003560 (SEQ ID NO: 838)CGGGCCCCGGGCGGGCGGGAGGGACGGGACGCGGUGCAGUGUUGUUUUUUCCCCCGCCAAUAUUGCACUCGUCCCGGCCUCCGGCCCCCCCGGCCC >hsa-mir-93 MI0000095 (SEQ ID NO: 839)CUGGGGGCUCCAAAGUGCUGUUCGUGCAGGUAGUGUGAUUACCCAACCUACUGCUGAGCUAGCACUUCCCGAGCCCCCGG >hsa-mir-95 MI0000097 (SEQ ID NO: 840)AACACAGUGGGCACUCAAUAAAUGUCUGUUGAAUUGAAAUGCGUUACAUUCAACGGGUAUUUAUUGAGCACCCACUCUGUG >hsa-mir-96 MI0000098 (SEQ ID NO: 841)UGGCCGAUUUUGGCACUAGCACAUUUUUGCUUGUGUCUCUCCGCUCUGAGCAAUCAUGUGCAGUGCCAAUAUGGGAAA >hsa-mir-98 MI0000100 (SEQ ID NO: 842)AGGAUUCUGCUCAUGCCAGGGUGAGGUAGUAAGUUGUAUUGUUGUGGGGUAGGGAUAUUAGGCCCCAAUUAGAAGAUAACUAUACAACUUACUACUUUCCCUGGUGUGUGGCAUAUUCA >hsa-mir-99a MI0000101 (SEQ ID NO: 843)CCCAUUGGCAUAAACCCGUAGAUCCGAUCUUGUGGUGAAGUGGACCGCACAAGCUCGCUUCUAUGGGUCUGUGUCAGUGUG >hsa-mir-99b MI0000746 (SEQ ID NO: 844)GGCACCCACCCGUAGAACCGACCUUGCGGGGCCUUCGCCGCACACAAGCUCGUGUCUGUGGGUCCGUGUC >hsa-mir-100 MI0000102 (SEQ ID NO: 845)CCUGUUGCCACAAACCCGUAGAUCCGAACUUGUGGUAUUAGUCCGCACAAGCUUGUAUCUAUAGGUAUGUGUCUGUUAGG >hsa-mir-101-1 MI0000103 (SEQ ID NO: 846)UGCCCUGGCUCAGUUAUCACAGUGCUGAUGCUGUCUAUUCUAAAGGUACAGUACUGUGAUAACUGAAGGAUGGCA >hsa-mir-101-2 MI0000739 (SEQ ID NO: 847)ACUGUCCUUUUUCGGUUAUCAUGGUACCGAUGCUGUAUAUCUGAAAGGUACAGUACUGUGAUAACUGAAGAAUGGUGGU >hsa-mir-103a-1 MI0000109 (SEQ ID NO: 848)UACUGCCCUCGGCUUCUUUACAGUGCUGCCUUGUUGCAUAUGGAUCAAGCAGCAUUGUACAGGGCUAUGAAGGCAUUG >hsa-mir-103a-2 MI0000108 (SEQ ID NO: 849)UUGUGCUUUCAGCUUCUUUACAGUGCUGCCUUGUAGCAUUCAGGUCAAGCAGCAUUGUACAGGGCUAUGAAAGAACCA >hsa-mir-103b-1 MI0007261 (SEQ ID NO: 850)UCAUAGCCCUGUACAAUGCUGCUUGAUCCAUAUGCAACAAGGCAGCACUGUAAAGAAGCCGA >hsa-mir-103b-2 MI0007262 (SEQ ID NO: 851)UCAUAGCCCUGUACAAUGCUGCUUGACCUGAAUGCUACAAGGCAGCACUGUAAAGAAGCUGA >hsa-mir-105-1 MI0000111 (SEQ ID NO: 852)UGUGCAUCGUGGUCAAAUGCUCAGACUCCUGUGGUGGCUGCUCAUGCACCACGGAUGUUUGAGCAUGUGCUACGGUGUCUA >hsa-mir-105-2 MI0000112 (SEQ ID NO: 853)UGUGCAUCGUGGUCAAAUGCUCAGACUCCUGUGGUGGCUGCUUAUGCACCACGGAUGUUUGAGCAUGUGCUAUGGUGUCUA >hsa-mir-106a MI0000113 (SEQ ID NO: 854)CCUUGGCCAUGUAAAAGUGCUUACAGUGCAGGUAGCUUUUUGAGAUCUACUGCAAUGUAAGCACUUCUUACAUUACCAUGG >hsa-mir-106b MI0000734 (SEQ ID NO: 855)CCUGCCGGGGCUAAAGUGCUGACAGUGCAGAUAGUGGUCCUCUCCGUGCUACCGCACUGUGGGUACUUGCUGCUCCAGCAGG >hsa-mir-107 MI0000114 (SEQ ID NO: 856)CUCUCUGCUUUCAGCUUCUUUACAGUGUUGCCUUGUGGCAUGGAGUUCAAGCAGCAUUGUACAGGGCUAUCAAAGCACAGA >hsa-mir-122 MI0000442 (SEQ ID NO: 857)CCUUAGCAGAGCUGUGGAGUGUGACAAUGGUGUUUGUGUCUAAACUAUCAAACGCCAUUAUCACACUAAAUAGCUACUGCUAGGC >hsa-mir-124-1 MI0000443 (SEQ ID NO: 858)AGGCCUCUCUCUCCGUGUUCACAGCGGACCUUGAUUUAAAUGUCCAUACAAUUAAGGCACGCGGUGAAUGCCAAGAAUGGGGCUG >hsa-mir-124-2 MI0000444 (SEQ ID NO: 859)AUCAAGAUUAGAGGCUCUGCUCUCCGUGUUCACAGCGGACCUUGAUUUAAUGUCAUACAAUUAAGGCACGCGGUGAAUGCCAAGAGCGGAGCCUACGGCUGCACUUGAA >hsa-mir-124-3 MI0000445 (SEQ ID NO: 860)UGAGGGCCCCUCUGCGUGUUCACAGCGGACCUUGAUUUAAUGUCUAUACAAUUAAGGCACGCGGUGAAUGCCAAGAGAGGCGCCUCC >hsa-mir-125a MI0000469 (SEQ ID NO: 861)UGCCAGUCUCUAGGUCCCUGAGACCCUUUAACCUGUGAGGACAUCCAGGGUCACAGGUGAGGUUCUUGGGAGCCUGGCGUCUGGCC >hsa-mir-125b-1 MI0000446 (SEQ ID NO: 862)UGCGCUCCUCUCAGUCCCUGAGACCCUAACUUGUGAUGUUUACCGUUUAAAUCCACGGGUUAGGCUCUUGGGAGCUGCGAGUCGUGCU >hsa-mir-125b-2 MI0000470 (SEQ ID NO: 863)ACCAGACUUUUCCUAGUCCCUGAGACCCUAACUUGUGAGGUAUUUUAGUAACAUCACAAGUCAGGCUCUUGGGACCUAGGCGGAGGGGA >hsa-mir-126 MI0000471 (SEQ ID NO: 864)CGCUGGCGACGGGACAUUAUUACUUUUGGUACGCGCUGUGACACUUCAAACUCGUACCGUGAGUAAUAAUGCGCCGUCCACGGCA >hsa-mir-127 MI0000472 (SEQ ID NO: 865)UGUGAUCACUGUCUCCAGCCUGCUGAAGCUCAGAGGGCUCUGAUUCAGAAAGAUCAUCGGAUCCGUCUGAGCUUGGCUGGUCGGAAGUCUCAUCAUC >hsa-mir-128-1 MI0000447 (SEQ ID NO: 866)UGAGCUGUUGGAUUCGGGGCCGUAGCACUGUCUGAGAGGUUUACAUUUCUCACAGUGAACCGGUCUCUUUUUCAGCUGCUUC >hsa-mir-128-2 MI0000727 (SEQ ID NO: 867)UGUGCAGUGGGAAGGGGGGCCGAUACACUGUACGAGAGUGAGUAGCAGGUCUCACAGUGAACCGGUCUCUUUCCCUACUGUGUC >hsa-mir-129-1 MI0000252 (SEQ ID NO: 868)GGAUCUUUUUGCGGUCUGGGCUUGCUGUUCCUCUCAACAGUAGUCAGGAAGCCCUUACCCCAAAAAGUAUCU >hsa-mir-129-2 MI0000473 (SEQ ID NO: 869)UGCCCUUCGCGAAUCUUUUUGCGGUCUGGGCUUGCUGUACAUAACUCAAUAGCCGGAAGCCCUUACCCCAAAAAGCAUUUGCGGAGGGCG >hsa-mir-130a MI0000448 (SEQ ID NO: 870)UGCUGCUGGCCAGAGCUCUUUUCACAUUGUGCUACUGUCUGCACCUGUCACUAGCAGUGCAAUGUUAAAAGGGCAUUGGCCGUGUAGUG >hsa-mir-130b MI0000748 (SEQ ID NO: 871)GGCCUGCCCGACACUCUUUCCCUGUUGCACUACUAUAGGCCGCUGGGAAGCAGUGCAAUGAUGAAAGGGCAUCGGUCAGGUC >hsa-mir-132 MI0000449 (SEQ ID NO: 872)CCGCCCCCGCGUCUCCAGGGCAACCGUGGCUUUCGAUUGUUACUGUGGGAACUGGAGGUAACAGUCUACAGCCAUGGUCGCCCCGCAGCACGCCCACGCGC >hsa-mir-133a-1 MI0000450 (SEQ ID NO: 873)ACAAUGCUUUGCUAGAGCUGGUAAAAUGGAACCAAAUCGCCUCUUCAAUGGAUUUGGUCCCCUUCAACCAGCUGUAGCUAUGCAUUGA >hsa-mir-133a-2 MI0000451 (SEQ ID NO: 874)GGGAGCCAAAUGCUUUGCUAGAGCUGGUAAAAUGGAACCAAAUCGACUGUCCAAUGGAUUUGGUCCCCUUCAACCAGCUGUAGCUGUGCAUUGAUGGCGCCG >hsa-mir-133b MI0000822 (SEQ ID NO: 875)CCUCAGAAGAAAGAUGCCCCCUGCUCUGGCUGGUCAAACGGAACCAAGUCCGUCUUCCUGAGAGGUUUGGUCCCCUUCAACCAGCUACAGCAGGGCUGGCAAUGCCCAGUCCUUGGAGA >hsa-mir-134 MI0000474 (SEQ ID NO: 876)CAGGGUGUGUGACUGGUUGACCAGAGGGGCAUGCACUGUGUUCACCCUGUGGGCCACCUAGUCACCAACCCUC >hsa-mir-135a-1 MI0000452 (SEQ ID NO: 877)AGGCCUCGCUGUUCUCUAUGGCUUUUUAUUCCUAUGUGAUUCUACUGCUCACUCAUAUAGGGAUUGGAGCCGUGGCGCACGGCGGGGACA >hsa-mir-135a-2 MI0000453 (SEQ ID NO: 878)AGAUAAAUUCACUCUAGUGCUUUAUGGCUUUUUAUUCCUAUGUGAUAGUAAUAAAGUCUCAUGUAGGGAUGGAAGCCAUGAAAUACAUUGUGAAAAAUCA >hsa-mir-135b MI0000810 (SEQ ID NO: 879)CACUCUGCUGUGGCCUAUGGCUUUUCAUUCCUAUGUGAUUGCUGUCCCAAACUCAUGUAGGGCUAAAAGCCAUGGGCUACAGUGAGGGGCGAGCUCC >hsa-mir-136 MI0000475 (SEQ ID NO: 880)UGAGCCCUCGGAGGACUCCAUUUGUUUUGAUGAUGGAUUCUUAUGCUCCAUCAUCGUCUCAAAUGAGUCUUCAGAGGGUUCU >hsa-mir-137 MI0000454 (SEQ ID NO: 881)GGUCCUCUGACUCUCUUCGGUGACGGGUAUUCUUGGGUGGAUAAUACGGAUUACGUUGUUAUUGCUUAAGAAUACGCGUAGUCGAGGAGAGUACCAGCGGCA >hsa-mir-138-1 MI0000476 (SEQ ID NO: 882)CCCUGGCAUGGUGUGGUGGGGCAGCUGGUGUUGUGAAUCAGGCCGUUGCCAAUCAGAGAACGGCUACUUCACAACACCAGGGCCACACCACACUACAGG >hsa-mir-138-2 MI0000455 (SEQ ID NO: 883)CGUUGCUGCAGCUGGUGUUGUGAAUCAGGCCGACGAGCAGCGCAUCCUCUUACCCGGCUAUUUCACGACACCAGGGUUGCAUCA >hsa-mir-139 MI0000261 (SEQ ID NO: 884)GUGUAUUCUACAGUGCACGUGUCUCCAGUGUGGCUCGGAGGCUGGAGACGCGGCCCUGUUGGAGUAAC >hsa-mir-140 MI0000456 (SEQ ID NO: 885)UGUGUCUCUCUCUGUGUCCUGCCAGUGGUUUUACCCUAUGGUAGGUUACGUCAUGCUGUUCUACCACAGGGUAGAACCACGGACAGGAUACCGGGGCACC >hsa-mir-141 MI0000457 (SEQ ID NO: 886)CGGCCGGCCCUGGGUCCAUCUUCCAGUACAGUGUUGGAUGGUCUAAUUGUGAAGCUCCUAACACUGUCUGGUAAAGAUGGCUCCCGGGUGGGUUC >hsa-mir-142 MI0000458 (SEQ ID NO: 887)GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG >hsa-mir-143 MI0000459 (SEQ ID NO: 888)GCGCAGCGCCCUGUCUCCCAGCCUGAGGUGCAGUGCUGCAUCUCUGGUCAGUUGGGAGUCUGAGAUGAAGCACUGUAGCUCAGGAAGAGAGAAGUUGUUCUGCAGC >hsa-mir-144 MI0000460 (SEQ ID NO: 889)UGGGGCCCUGGCUGGGAUAUCAUCAUAUACUGUAAGUUUGCGAUGAGACACUACAGUAUAGAUGAUGUACUAGUCCGGGCACCCCC >hsa-mir-145 MI0000461 (SEQ ID NO: 890)CACCUUGUCCUCACGGUCCAGUUUUCCCAGGAAUCCCUUAGAUGCUAAGAUGGGGAUUCCUGGAAAUACUGUUCUUGAGGUCAUGGUU >hsa-mir-146a MI0000477 (SEQ ID NO: 891)CCGAUGUGUAUCCUCAGCUUUGAGAACUGAAUUCCAUGGGUUGUGUCAGUGUCAGACCUCUGAAAUUCAGUUCUUCAGCUGGGAUAUCUCUGUCAUCGU >hsa-mir-146b MI0003129 (SEQ ID NO: 892)CCUGGCACUGAGAACUGAAUUCCAUAGGCUGUGAGCUCUAGCAAUGCCCUGUGGACUCAGUUCUGGUGCCCGG >hsa-mir-147a MI0000262 (SEQ ID NO: 893)AAUCUAAAGACAACAUUUCUGCACACACACCAGACUAUGGAAGCCAGUGUGUGGAAAUGCUUCUGCUAGAUU >hsa-mir-147b MI0005544 (SEQ ID NO: 894)UAUAAAUCUAGUGGAAACAUUUCUGCACAAACUAGAUUCUGGACACCAGUGUGCGGAAAUGCUUCUGCUACAUUUUUAGG >hsa-mir-148a MI0000253 (SEQ ID NO: 895)GAGGCAAAGUUCUGAGACACUCCGACUCUGAGUAUGAUAGAAGUCAGUGCACUACAGAACUUUGUCUC >hsa-mir-148b MI0000811 (SEQ ID NO: 896)CAAGCACGAUUAGCAUUUGAGGUGAAGUUCUGUUAUACACUCAGGCUGUGGCUCUCUGAAAGUCAGUGCAUCACAGAACUUUGUCUCGAAAGCUUUCUA >hsa-mir-149 MI0000478 (SEQ ID NO: 897)GCCGGCGCCCGAGCUCUGGCUCCGUGUCUUCACUCCCGUGCUUGUCCGAGGAGGGAGGGAGGGACGGGGGCUGUGCUGGGGCAGCUGGA >hsa-mir-150 MI0000479 (SEQ ID NO: 898)CUCCCCAUGGCCCUGUCUCCCAACCCUUGUACCAGUGCUGGGCUCAGACCCUGGUACAGGCCUGGGGGACAGGGACCUGGGGAC >hsa-mir-151a MI0000809 (SEQ ID NO: 899)UUUCCUGCCCUCGAGGAGCUCACAGUCUAGUAUGUCUCAUCCCCUACUAGACUGAAGCUCCUUGAGGACAGGGAUGGUCAUACUCACCUC >hsa-mir-151b MI0003772 (SEQ ID NO: 900)ACCUCUGAUGUGUCAGUCUCUCUUCAGGGCUCCCGAGACACAGAAACAGACACCUGCCCUCGAGGAGCUCACAGUCUAGACAAACAAACCCAGGGU >hsa-mir-152 MI0000462 (SEQ ID NO: 901)UGUCCCCCCCGGCCCAGGUUCUGUGAUACACUCCGACUCGGGCUCUGGAGCAGUCAGUGCAUGACAGAACUUGGGCCCGGAAGGACC >hsa-mir-153-1 MI0000463 (SEQ ID NO: 902)CUCACAGCUGCCAGUGUCAUUUUUGUGAUCUGCAGCUAGUAUUCUCACUCCAGUUGCAUAGUCACAAAAGUGAUCAUUGGCAGGUGUGGC >hsa-mir-153-2 MI0000464 (SEQ ID NO: 903)AGCGGUGGCCAGUGUCAUUUUUGUGAUGUUGCAGCUAGUAAUAUGAGCCCAGUUGCAUAGUCACAAAAGUGAUCAUUGGAAACUGUG >hsa-mir-154 MI0000480 (SEQ ID NO: 904)GUGGUACUUGAAGAUAGGUUAUCCGUGUUGCCUUCGCUUUAUUUGUGACGAAUCAUACACGGUUGACCUAUUUUUCAGUACCAA >hsa-mir-155 MI0000681 (SEQ ID NO: 905)CUGUUAAUGCUAAUCGUGAUAGGGGUUUUUGCCUCCAACUGACUCCUACAUAUUAGCAUUAACAG >hsa-mir-181a-1 MI0000289 (SEQ ID NO: 906)UGAGUUUUGAGGUUGCUUCAGUGAACAUUCAACGCUGUCGGUGAGUUUGGAAUUAAAAUCAAAACCAUCGACCGUUGAUUGUACCCUAUGGCUAACCAUCAUCUACUCCA >hsa-mir-181a-2 MI0000269 (SEQ ID NO: 907)AGAAGGGCUAUCAGGCCAGCCUUCAGAGGACUCCAAGGAACAUUCAACGCUGUCGGUGAGUUUGGGAUUUGAAAAAACCACUGACCGUUGACUGUACCUUGGGGUCCUUA >hsa-mir-181b-1 MI0000270 (SEQ ID NO: 908)CCUGUGCAGAGAUUAUUUUUUAAAAGGUCACAAUCAACAUUCAUUGCUGUCGGUGGGUUGAACUGUGUGGACAAGCUCACUGAACAAUGAAUGCAACUGUGGCCCCGCUU >hsa-mir-181b-2 MI0000683 (SEQ ID NO: 909)CUGAUGGCUGCACUCAACAUUCAUUGCUGUCGGUGGGUUUGAGUCUGAAUCAACUCACUGAUCAAUGAAUGCAAACUGCGGACCAAACA >hsa-mir-181c MI0000271 (SEQ ID NO: 910)CGGAAAAUUUGCCAAGGGUUUGGGGGAACAUUCAACCUGUCGGUGAGUUUGGGCAGCUCAGGCAAACCAUCGACCGUUGAGUGGACCCUGAGGCCUGGAAUUGCCAUCCU >hsa-mir-181d MI0003139 (SEQ ID NO: 911)GUCCCCUCCCCUAGGCCACAGCCGAGGUCACAAUCAACAUUCAUUGUUGUCGGUGGGUUGUGAGGACUGAGGCCAGACCCACCGGGGGAUGAAUGUCACUGUGGCUGGGCCAGACACGGCUUAAGGGGAAUGGGGAC >hsa-mir-182 MI0000272 (SEQ ID NO: 912)GAGCUGCUUGCCUCCCCCCGUUUUUGGCAAUGGUAGAACUCACACUGGUGAGGUAACAGGAUCCGGUGGUUCUAGACUUGCCAACUAUGGGGCGAGGACUCAGCCGGCAC >hsa-mir-183 MI0000273 (SEQ ID NO: 913)CCGCAGAGUGUGACUCCUGUUCUGUGUAUGGCACUGGUAGAAUUCACUGUGAACAGUCUCAGUCAGUGAAUUACCGAAGGGCCAUAAACAGAGCAGAGACAGAUCCACGA >hsa-mir-184 MI0000481 (SEQ ID NO: 914)CCAGUCACGUCCCCUUAUCACUUUUCCAGCCCAGCUUUGUGACUGUAAGUGUUGGACGGAGAACUGAUAAGGGUAGGUGAUUGA >hsa-mir-185 MI0000482 (SEQ ID NO: 915)AGGGGGCGAGGGAUUGGAGAGAAAGGCAGUUCCUGAUGGUCCCCUCCCCAGGGGCUGGCUUUCCUCUGGUCCUUCCCUCCCA >hsa-mir-186 MI0000483 (SEQ ID NO: 916)UGCUUGUAACUUUCCAAAGAAUUCUCCUUUUGGGCUUUCUGGUUUUAUUUUAAGCCCAAAGGUGAAUUUUUUGGGAAGUUUGAGCU >hsa-mir-187 MI0000274 (SEQ ID NO: 917)GGUCGGGCUCACCAUGACACAGUGUGAGACCUCGGGCUACAACACAGGACCCGGGCGCUGCUCUGACCCCUCGUGUCUUGUGUUGCAGCCGGAGGGACGCAGGUCCGCA >hsa-mir-188 MI0000484 (SEQ ID NO: 918)UGCUCCCUCUCUCACAUCCCUUGCAUGGUGGAGGGUGAGCUUUCUGAAAACCCCUCCCACAUGCAGGGUUUGCAGGAUGGCGAGCC >hsa-mir-190a MI0000486 (SEQ ID NO: 919)UGCAGGCCUCUGUGUGAUAUGUUUGAUAUAUUAGGUUGUUAUUUAAUCCAACUAUAUAUCAAACAUAUUCCUACAGUGUCUUGCC >hsa-mir-190b MI0005545 (SEQ ID NO: 920)UGCUUCUGUGUGAUAUGUUUGAUAUUGGGUUGUUUAAUUAGGAACCAACUAAAUGUCAAACAUAUUCUUACAGCAGCAG >hsa-mir-191 MI0000465 (SEQ ID NO: 921)CGGCUGGACAGCGGGCAACGGAAUCCCAAAAGCAGCUGUUGUCUCCAGAGCAUUCCAGCUGCGCUUGGAUUUCGUCCCCUGCUCUCCUGCCU >hsa-mir-192 MI0000234 (SEQ ID NO: 922)GCCGAGACCGAGUGCACAGGGCUCUGACCUAUGAAUUGACAGCCAGUGCUCUCGUCUCCCCUCUGGCUGCCAAUUCCAUAGGUCACAGGUAUGUUCGCCUCAAUGCCAGC >hsa-mir-193a MI0000487 (SEQ ID NO: 923)CGAGGAUGGGAGCUGAGGGCUGGGUCUUUGCGGGCGAGAUGAGGGUGUCGGAUCAACUGGCCUACAAAGUCCCAGUUCUCGGCCCCCG >hsa-mir-193b MI0003137 (SEQ ID NO: 924)GUGGUCUCAGAAUCGGGGUUUUGAGGGCGAGAUGAGUUUAUGUUUUAUCCAACUGGCCCUCAAAGUCCCGCUUUUGGGGUCAU >hsa-mir-194-1 MI0000488 (SEQ ID NO: 925)AUGGUGUUAUCAAGUGUAACAGCAACUCCAUGUGGACUGUGUACCAAUUUCCAGUGGAGAUGCUGUUACUUUUGAUGGUUACCAA >hsa-mir-194-2 MI0000732 (SEQ ID NO: 926)UGGUUCCCGCCCCCUGUAACAGCAACUCCAUGUGGAAGUGCCCACUGGUUCCAGUGGGGCUGCUGUUAUCUGGGGCGAGGGCCAG >hsa-mir-195 MI0000489 (SEQ ID NO: 927)AGCUUCCCUGGCUCUAGCAGCACAGAAAUAUUGGCACAGGGAAGCGAGUCUGCCAAUAUUGGCUGUGCUGCUCCAGGCAGGGUGGUG >hsa-mir-196a-1 MI0000238 (SEQ ID NO: 928)GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGGGUUUCUGAACACAACAACAUUAAACCACCCGAUUCAC >hsa-mir-196a-2 MI0000279 (SEQ ID NO: 929)UGCUCGCUCAGCUGAUCUGUGGCUUAGGUAGUUUCAUGUUGUUGGGAUUGAGUUUUGAACUCGGCAACAAGAAACUGCCUGAGUUACAUCAGUCGGUUUUCGUCGAGGGC >hsa-mir-196b MI0001150 (SEQ ID NO: 930)ACUGGUCGGUGAUUUAGGUAGUUUCCUGUUGUUGGGAUCCACCUUUCUCUCGACAGCACGACACUGCCUUCAUUACUUCAGUUG >hsa-mir-197 MI0000239 (SEQ ID NO: 931)GGCUGUGCCGGGUAGAGAGGGCAGUGGGAGGUAAGAGCUCUUCACCCUUCACCACCUUCUCCACCCAGCAUGGCC >hsa-mir-198 MI0000240 (SEQ ID NO: 932)UCAUUGGUCCAGAGGGGAGAUAGGUUCCUGUGAUUUUUCCUUCUUCUCUAUAGAAUAAAUGA >hsa-mir-199a-1 MI0000242 (SEQ ID NO: 933)GCCAACCCAGUGUUCAGACUACCUGUUCAGGAGGCUCUCAAUGUGUACAGUAGUCUGCACAUUGGUUAGGC >hsa-mir-199a-2 MI0000281 (SEQ ID NO: 934)AGGAAGCUUCUGGAGAUCCUGCUCCGUCGCCCCAGUGUUCAGACUACCUGUUCAGGACAAUGCCGUUGUACAGUAGUCUGCACAUUGGUUAGACUGGGCAAGGGAGAGCA >hsa-mir-199b MI0000282 (SEQ ID NO: 935)CCAGAGGACACCUCCACUCCGUCUACCCAGUGUUUAGACUAUCUGUUCAGGACUCCCAAAUUGUACAGUAGUCUGCACAUUGGUUAGGCUGGGCUGGGUUAGACCCUCGG >hsa-mir-200a MI0000737 (SEQ ID NO: 936)CCGGGCCCCUGUGAGCAUCUUACCGGACAGUGCUGGAUUUCCCAGCUUGACUCUAACACUGUCUGGUAACGAUGUUCAAAGGUGACCCGC >hsa-mir-200b MI0000342 (SEQ ID NO: 937)CCAGCUCGGGCAGCCGUGGCCAUCUUACUGGGCAGCAUUGGAUGGAGUCAGGUCUCUAAUACUGCCUGGUAAUGAUGACGGCGGAGCCCUGCACG >hsa-mir-200c MI0000650 (SEQ ID NO: 938)CCCUCGUCUUACCCAGCAGUGUUUGGGUGCGGUUGGGAGUCUCUAAUACUGCCGGGUAAUGAUGGAGG >hsa-mir-202 MI0003130 (SEQ ID NO: 939)CGCCUCAGAGCCGCCCGCCGUUCCUUUUUCCUAUGCAUAUACUUCUUUGAGGAUCUGGCCUAAAGAGGUAUAGGGCAUGGGAAAACGGGGCGGUCGGGUCCUCCCCAGCG >hsa-mir-203 MI0000283 (SEQ ID NO: 940)GUGUUGGGGACUCGCGCGCUGGGUCCAGUGGUUCUUAACAGUUCAACAGUUCUGUAGCGCAAUUGUGAAAUGUUUAGGACCACUAGACCCGGCGGGCGCGGCGACAGCGA >hsa-mir-204 MI0000284 (SEQ ID NO: 941)GGCUACAGUCUUUCUUCAUGUGACUCGUGGACUUCCCUUUGUCAUCCUAUGCCUGAGAAUAUAUGAAGGAGGCUGGGAAGGCAAAGGGACGUUCAAUUGUCAUCACUGGC >hsa-mir-205 MI0000285 (SEQ ID NO: 942)AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACA >hsa-mir-206 MI0000490 (SEQ ID NO: 943)UGCUUCCCGAGGCCACAUGCUUCUUUAUAUCCCCAUAUGGAUUACUUUGCUAUGGAAUGUAAGGAAGUGUGUGGUUUCGGCAAGUG >hsa-mir-208a MI0000251 (SEQ ID NO: 944)UGACGGGCGAGCUUUUGGCCCGGGUUAUACCUGAUGCUCACGUAUAAGACGAGCAAAAAGCUUGUUGGUCA >hsa-mir-208b MI0005570 (SEQ ID NO: 945)CCUCUCAGGGAAGCUUUUUGCUCGAAUUAUGUUUCUGAUCCGAAUAUAAGACGAACAAAAGGUUUGUCUGAGGGCAG >hsa-mir-210 MI0000286 (SEQ ID NO: 946)ACCCGGCAGUGCCUCCAGGCGCAGGGCAGCCCCUGCCCACCGCACACUGCGCUGCCCCAGACCCACUGUGCGUGUGACAGCGGCUGAUCUGUGCCUGGGCAGCGCGACCC >hsa-mir-211 MI0000287 (SEQ ID NO: 947)UCACCUGGCCAUGUGACUUGUGGGCUUCCCUUUGUCAUCCUUCGCCUAGGGCUCUGAGCAGGGCAGGGACAGCAAAGGGGUGCUCAGUUGUCACUUCCCACAGCACGGAG >hsa-mir-212 MI0000288 (SEQ ID NO: 948)CGGGGCACCCCGCCCGGACAGCGCGCCGGCACCUUGGCUCUAGACUGCUUACUGCCCGGGCCGCCCUCAGUAACAGUCUCCAGUCACGGCCACCGACGCCUGGCCCCGCC >hsa-mir-214 MI0000290 (SEQ ID NO: 949)GGCCUGGCUGGACAGAGUUGUCAUGUGUCUGCCUGUCUACACUUGCUGUGCAGAACAUCCGCUCACCUGUACAGCAGGCACAGACAGGCAGUCACAUGACAACCCAGCCU >hsa-mir-215 MI0000291 (SEQ ID NO: 950)AUCAUUCAGAAAUGGUAUACAGGAAAAUGACCUAUGAAUUGACAGACAAUAUAGCUGAGUUUGUCUGUCAUUUCUUUAGGCCAAUAUUCUGUAUGACUGUGCUACUUCAA >hsa-mir-216a MI0000292 (SEQ ID NO: 951)GAUGGCUGUGAGUUGGCUUAAUCUCAGCUGGCAACUGUGAGAUGUUCAUACAAUCCCUCACAGUGGUCUCUGGGAUUAUGCUAAACAGAGCAAUUUCCUAGCCCUCACGA >hsa-mir-216b MI0005569 (SEQ ID NO: 952)GCAGACUGGAAAAUCUCUGCAGGCAAAUGUGAUGUCACUGAGGAAAUCACACACUUACCCGUAGAGAUUCUACAGUCUGACA >hsa-mir-217 MI0000293 (SEQ ID NO: 953)AGUAUAAUUAUUACAUAGUUUUUGAUGUCGCAGAUACUGCAUCAGGAACUGAUUGGAUAAGAAUCAGUCACCAUCAGUUCCUAAUGCAUUGCCUUCAGCAUCUAAACAAG >hsa-mir-218-1 MI0000294 (SEQ ID NO: 954)GUGAUAAUGUAGCGAGAUUUUCUGUUGUGCUUGAUCUAACCAUGUGGUUGCGAGGUAUGAGUAAAACAUGGUUCCGUCAAGCACCAUGGAACGUCACGCAGCUUUCUACA >hsa-mir-218-2 MI0000295 (SEQ ID NO: 955)GACCAGUCGCUGCGGGGCUUUCCUUUGUGCUUGAUCUAACCAUGUGGUGGAACGAUGGAAACGGAACAUGGUUCUGUCAAGCACCGCGGAAAGCACCGUGCUCUCCUGCA >hsa-mir-219-1 MI0000296 (SEQ ID NO: 956)CCGCCCCGGGCCGCGGCUCCUGAUUGUCCAAACGCAAUUCUCGAGUCUAUGGCUCCGGCCGAGAGUUGAGUCUGGACGUCCCGAGCCGCCGCCCCCAAACCUCGAGCGGG >hsa-mir-219-2 MI0000740 (SEQ ID NO: 957)ACUCAGGGGCUUCGCCACUGAUUGUCCAAACGCAAUUCUUGUACGAGUCUGCGGCCAACCGAGAAUUGUGGCUGGACAUCUGUGGCUGAGCUCCGGG >hsa-mir-221 MI0000298 (SEQ ID NO: 958)UGAACAUCCAGGUCUGGGGCAUGAACCUGGCAUACAAUGUAGAUUUCUGUGUUCGUUAGGCAACAGCUACAUUGUCUGCUGGGUUUCAGGCUACCUGGAAACAUGUUCUC >hsa-mir-222 MI0000299 (SEQ ID NO: 959)GCUGCUGGAAGGUGUAGGUACCCUCAAUGGCUCAGUAGCCAGUGUAGAUCCUGUCUUUCGUAAUCAGCAGCUACAUCUGGCUACUGGGUCUCUGAUGGCAUCUUCUAGCU >hsa-mir-223 MI0000300 (SEQ ID NO: 960)CCUGGCCUCCUGCAGUGCCACGCUCCGUGUAUUUGACAAGCUGAGUUGGACACUCCAUGUGGUAGAGUGUCAGUUUGUCAAAUACCCCAAGUGCGGCACAUGCUUACCAG >hsa-mir-224 MI0000301 (SEQ ID NO: 961)GGGCUUUCAAGUCACUAGUGGUUCCGUUUAGUAGAUGAUUGUGCAUUGUUUCAAAAUGGUGCCCUAGUGACUACAAAGCCC >hsa-mir-296 MI0000747 (SEQ ID NO: 962)AGGACCCUUCCAGAGGGCCCCCCCUCAAUCCUGUUGUGCCUAAUUCAGAGGGUUGGGUGGAGGCUCUCCUGAAGGGCUCU >hsa-mir-297 MI0005775 (SEQ ID NO: 963)UGUAUGUAUGUGUGCAUGUGCAUGUAUGUGUAUAUACAUAUAUAUGUAUUAUGUACUCAUAUAUCA >hsa-mir-298 MI0005523 (SEQ ID NO: 964)UCAGGUCUUCAGCAGAAGCAGGGAGGUUCUCCCAGUGGUUUUCCUUGACUGUGAGGAACUAGCCUGCUGCUUUGCUCAGGAGUGAGCU >hsa-mir-299 MI0000744 (SEQ ID NO: 965)AAGAAAUGGUUUACCGUCCCACAUACAUUUUGAAUAUGUAUGUGGGAUGGUAAACCGCUUCUU >hsa-mir-300 MI0005525 (SEQ ID NO: 966)UGCUACUUGAAGAGAGGUAAUCCUUCACGCAUUUGCUUUACUUGCAAUGAUUAUACAAGGGCAGACUCUCUCUGGGGAGCAAA >hsa-mir-301a MI0000745 (SEQ ID NO: 967)ACUGCUAACGAAUGCUCUGACUUUAUUGCACUACUGUACUUUACAGCUAGCAGUGCAAUAGUAUUGUCAAAGCAUCUGAAAGCAGG >hsa-mir-301b MI0005568 (SEQ ID NO: 968)GCCGCAGGUGCUCUGACGAGGUUGCACUACUGUGCUCUGAGAAGCAGUGCAAUGAUAUUGUCAAAGCAUCUGGGACCA >hsa-mir-302a MI0000738 (SEQ ID NO: 969)CCACCACUUAAACGUGGAUGUACUUGCUUUGAAACUAAAGAAGUAAGUGCUUCCAUGUUUUGGUGAUGG >hsa-mir-302b MI0000772 (SEQ ID NO: 970)GCUCCCUUCAACUUUAACAUGGAAGUGCUUUCUGUGACUUUAAAAGUAAGUGCUUCCAUGUUUUAGUAGGAGU >hsa-mir-302c MI0000773 (SEQ ID NO: 971)CCUUUGCUUUAACAUGGGGGUACCUGCUGUGUGAAACAAAAGUAAGUGCUUCCAUGUUUCAGUGGAGG >hsa-mir-302d MI0000774 (SEQ ID NO: 972)CCUCUACUUUAACAUGGAGGCACUUGCUGUGACAUGACAAAAAUAAGUGCUUCCAUGUUUGAGUGUGG >hsa-mir-302e MI0006417 (SEQ ID NO: 973)UUGGGUAAGUGCUUCCAUGCUUCAGUUUCCUUACUGGUAAGAUGGAUGUAGUAAUAGCACCUACCUUAUAGA >hsa-mir-302f MI0006418 (SEQ ID NO: 974)UCUGUGUAAACCUGGCAAUUUUCACUUAAUUGCUUCCAUGUUUAUAAAAGA >hsa-mir-320a MI0000542 (SEQ ID NO: 975)GCUUCGCUCCCCUCCGCCUUCUCUUCCCGGUUCUUCCCGGAGUCGGGAAAAGCUGGGUUGAGAGGGCGAAAAAGGAUGAGGU >hsa-mir-320b-1 MI0003776 (SEQ ID NO: 976)AAUUAAUCCCUCUCUUUCUAGUUCUUCCUAGAGUGAGGAAAAGCUGGGUUGAGAGGGCAAACAAAUUAACUAAUUAAUU >hsa-mir-320b-2 MI0003839 (SEQ ID NO: 977)UGUUAUUUUUUGUCUUCUACCUAAGAAUUCUGUCUCUUAGGCUUUCUCUUCCCAGAUUUCCCAAAGUUGGGAAAAGCUGGGUUGAGAGGGCAAAAGGAAAAAAAAAGAAUUCUGUCUCUGACAUAAUUAGAUAGGGAA >hsa-mir-320c-1 MI0003778 (SEQ ID NO: 978)UUUGCAUUAAAAAUGAGGCCUUCUCUUCCCAGUUCUUCCCAGAGUCAGGAAAAGCUGGGUUGAGAGGGUAGAAAAAAAAUGAUGUAGG >hsa-mir-320c-2 MI0008191 (SEQ ID NO: 979)CUUCUCUUUCCAGUUCUUCCCAGAAUUGGGAAAAGCUGGGUUGAGAGGGU >hsa-mir-320d-1 MI0008190 (SEQ ID NO: 980)UUCUCGUCCCAGUUCUUCCCAAAGUUGAGAAAAGCUGGGUUGAGAGGA >hsa-mir-320d-2 MI0008192 (SEQ ID NO: 981)UUCUCUUCCCAGUUCUUCUUGGAGUCAGGAAAAGCUGGGUUGAGAGGA >hsa-mir-320e MI0014234 (SEQ ID NO: 982)GCCUUCUCUUCCCAGUUCUUCCUGGAGUCGGGGAAAAGCUGGGUUGAGAAGGU >hsa-mir-323a MI0000807 (SEQ ID NO: 983)UUGGUACUUGGAGAGAGGUGGUCCGUGGCGCGUUCGCUUUAUUUAUGGCGCACAUUACACGGUCGACCUCUUUGCAGUAUCUAAUC >hsa-mir-323b MI0014206 (SEQ ID NO: 984)UGGUACUCGGAGGGAGGUUGUCCGUGGUGAGUUCGCAUUAUUUAAUGAUGCCCAAUACACGGUCGACCUCUUUUCGGUAUCA >hsa-mir-324 MI0000813 (SEQ ID NO: 985)CUGACUAUGCCUCCCCGCAUCCCCUAGGGCAUUGGUGUAAAGCUGGAGACCCACUGCCCCAGGUGCUGCUGGGGGUUGUAGUC >hsa-mir-325 MI0000824 (SEQ ID NO: 986)AUACAGUGCUUGGUUCCUAGUAGGUGUCCAGUAAGUGUUUGUGACAUAAUUUGUUUAUUGAGGACCUCCUAUCAAUCAAGCACUGUGCUAGGCUCUGG >hsa-mir-326 MI0000808 (SEQ ID NO: 987)CUCAUCUGUCUGUUGGGCUGGAGGCAGGGCCUUUGUGAAGGCGGGUGGUGCUCAGAUCGCCUCUGGGCCCUUCCUCCAGCCCCGAGGCGGAUUCA >hsa-mir-328 MI0000804 (SEQ ID NO: 988)UGGAGUGGGGGGGCAGGAGGGGCUCAGGGAGAAAGUGCAUACAGCCCCUGGCCCUCUCUGCCCUUCCGUCCCCUG >hsa-mir-329-1 MI0001725 (SEQ ID NO: 989)GGUACCUGAAGAGAGGUUUUCUGGGUUUCUGUUUCUUUAAUGAGGACGAAACACACCUGGUUAACCUCUUUUCCAGUAUC >hsa-mir-329-2 MI0001726 (SEQ ID NO: 990)GUGGUACCUGAAGAGAGGUUUUCUGGGUUUCUGUUUCUUUAUUGAGGACGAAACACACCUGGUUAACCUCUUUUCCAGUAUCAA >hsa-mir-330 MI0000803 (SEQ ID NO: 991)CUUUGGCGAUCACUGCCUCUCUGGGCCUGUGUCUUAGGCUCUGCAAGAUCAACCGAGCAAAGCACACGGCCUGCAGAGAGGCAGCGCUCUGCCC >hsa-mir-331 MI0000812 (SEQ ID NO: 992)GAGUUUGGUUUUGUUUGGGUUUGUUCUAGGUAUGGUCCCAGGGAUCCCAGAUCAAACCAGGCCCCUGGGCCUAUCCUAGAACCAACCUAAGCUC >hsa-mir-335 MI0000816 (SEQ ID NO: 993)UGUUUUGAGCGGGGGUCAAGAGCAAUAACGAAAAAUGUUUGUCAUAAACCGUUUUUCAUUAUUGCUCCUGACCUCCUCUCAUUUGCUAUAUUCA >hsa-mir-337 MI0000806 (SEQ ID NO: 994)GUAGUCAGUAGUUGGGGGGUGGGAACGGCUUCAUACAGGAGUUGAUGCACAGUUAUCCAGCUCCUAUAUGAUGCCUUUCUUCAUCCCCUUCAA >hsa-mir-338 MI0000814 (SEQ ID NO: 995)UCUCCAACAAUAUCCUGGUGCUGAGUGAUGACUCAGGCGACUCCAGCAUCAGUGAUUUUGUUGAAGA >hsa-mir-339 MI0000815 (SEQ ID NO: 996)CGGGGCGGCCGCUCUCCCUGUCCUCCAGGAGCUCACGUGUGCCUGCCUGUGAGCGCCUCGACGACAGAGCCGGCGCCUGCCCCAGUGUCUGCGC >hsa-mir-340 MI0000802 (SEQ ID NO: 997)UUGUACCUGGUGUGAUUAUAAAGCAAUGAGACUGAUUGUCAUAUGUCGUUUGUGGGAUCCGUCUCAGUUACUUUAUAGCCAUACCUGGUAUCUUA >hsa-mir-342 MI0000805 (SEQ ID NO: 998)GAAACUGGGCUCAAGGUGAGGGGUGCUAUCUGUGAUUGAGGGACAUGGUUAAUGGAAUUGUCUCACACAGAAAUCGCACCCGUCACCUUGGCCUACUUA >hsa-mir-345 MI0000825 (SEQ ID NO: 999)ACCCAAACCCUAGGUCUGCUGACUCCUAGUCCAGGGCUCGUGAUGGCUGGUGGGCCCUGAACGAGGGGUCUGGAGGCCUGGGUUUGAAUAUCGACAGC >hsa-mir-346 MI0000826 (SEQ ID NO: 1000)GGUCUCUGUGUUGGGCGUCUGUCUGCCCGCAUGCCUGCCUCUCUGUUGCUCUGAAGGAGGCAGGGGCUGGGCCUGCAGCUGCCUGGGCAGAGCGG >hsa-mir-361 MI0000760 (SEQ ID NO: 1001)GGAGCUUAUCAGAAUCUCCAGGGGUACUUUAUAAUUUCAAAAAGUCCCCCAGGUGUGAUUCUGAUUUGCUUC >hsa-mir-362 MI0000762 (SEQ ID NO: 1002)CUUGAAUCCUUGGAACCUAGGUGUGAGUGCUAUUUCAGUGCAACACACCUAUUCAAGGAUUCAAA >hsa-mir-363 MI0000764 (SEQ ID NO: 1003)UGUUGUCGGGUGGAUCACGAUGCAAUUUUGAUGAGUAUCAUAGGAGAAAAAUUGCACGGUAUCCAUCUGUAAACC >hsa-mir-365a MI0000767 (SEQ ID NO: 1004)ACCGCAGGGAAAAUGAGGGACUUUUGGGGGCAGAUGUGUUUCCAUUCCACUAUCAUAAUGCCCCUAAAAAUCCUUAUUGCUCUUGCA >hsa-mir-365b MI0000769 (SEQ ID NO: 1005)AGAGUGUUCAAGGACAGCAAGAAAAAUGAGGGACUUUCAGGGGCAGCUGUGUUUUCUGACUCAGUCAUAAUGCCCCUAAAAAUCCUUAUUGUUCUUGCAGUGUGCAUCGGG >hsa-mir-367 MI0000775 (SEQ ID NO: 1006)CCAUUACUGUUGCUAAUAUGCAACUCUGUUGAAUAUAAAUUGGAAUUGCACUUUAGCAAUGGUGAUGG >hsa-mir-369 MI0000777 (SEQ ID NO: 1007)UUGAAGGGAGAUCGACCGUGUUAUAUUCGCUUUAUUGACUUCGAAUAAUACAUGGUUGAUCUUUUCUCAG >hsa-mir-370 MI0000778 (SEQ ID NO: 1008)AGACAGAGAAGCCAGGUCACGUCUCUGCAGUUACACAGCUCACGAGUGCCUGCUGGGGUGGAACCUGGUCUGUCU >hsa-mir-371a MI0000779 (SEQ ID NO: 1009)GUGGCACUCAAACUGUGGGGGCACUUUCUGCUCUCUGGUGAAAGUGCCGCCAUCUUUUGAGUGUUAC >hsa-mir-371b MI0017393 (SEQ ID NO: 1010)GGUAACACUCAAAAGAUGGCGGCACUUUCACCAGAGAGCAGAAAGUGCCCCCACAGUUUGAGUGCC >hsa-mir-372 MI0000780 (SEQ ID NO: 1011)GUGGGCCUCAAAUGUGGAGCACUAUUCUGAUGUCCAAGUGGAAAGUGCUGCGACAUUUGAGCGUCAC >hsa-mir-373 MI0000781 (SEQ ID NO: 1012)GGGAUACUCAAAAUGGGGGCGCUUUCCUUUUUGUCUGUACUGGGAAGUGCUUCGAUUUUGGGGUGUCCC >hsa-mir-374a MI0000782 (SEQ ID NO: 1013)UACAUCGGCCAUUAUAAUACAACCUGAUAAGUGUUAUAGCACUUAUCAGAUUGUAUUGUAAUUGUCUGUGUA >hsa-mir-374b MI0005566 (SEQ ID NO: 1014)ACUCGGAUGGAUAUAAUACAACCUGCUAAGUGUCCUAGCACUUAGCAGGUUGUAUUAUCAUUGUCCGUGUCU >hsa-mir-374c MI0016684 (SEQ ID NO: 1015)ACACGGACAAUGAUAAUACAACCUGCUAAGUGCUAGGACACUUAGCAGGUUGUAUUAUAUCCAUCCGAGU >hsa-mir-375 MI0000783 (SEQ ID NO: 1016)CCCCGCGACGAGCCCCUCGCACAAACCGGACCUGAGCGUUUUGUUCGUUCGGCUCGCGUGAGGC >hsa-mir-376a-1 MI0000784 (SEQ ID NO: 1017)UAAAAGGUAGAUUCUCCUUCUAUGAGUACAUUAUUUAUGAUUAAUCAUAGAGGAAAAUCCACGUUUUC >hsa-mir-376a-2 MI0003529 (SEQ ID NO: 1018)GGUAUUUAAAAGGUAGAUUUUCCUUCUAUGGUUACGUGUUUGAUGGUUAAUCAUAGAGGAAAAUCCACGUUUUCAGUAUC >hsa-mir-376b MI0002466 (SEQ ID NO: 1019)CAGUCCUUCUUUGGUAUUUAAAACGUGGAUAUUCCUUCUAUGUUUACGUGAUUCCUGGUUAAUCAUAGAGGAAAAUCCAUGUUUUCAGUAUCAAAUGCUG >hsa-mir-376c MI0000776 (SEQ ID NO: 1020)AAAAGGUGGAUAUUCCUUCUAUGUUUAUGUUAUUUAUGGUUAAACAUAGAGGAAAUUCCACGUUUU >hsa-mir-377 MI0000785 (SEQ ID NO: 1021)UUGAGCAGAGGUUGCCCUUGGUGAAUUCGCUUUAUUUAUGUUGAAUCACACAAAGGCAACUUUUGUUUG >hsa-mir-378a MI0000786 (SEQ ID NO: 1022)AGGGCUCCUGACUCCAGGUCCUGUGUGUUACCUAGAAAUAGCACUGGACUUGGAGUCAGAAGGCCU >hsa-mir-378b MI0014154 (SEQ ID NO: 1023)GGUCAUUGAGUCUUCAAGGCUAGUGGAAAGAGCACUGGACUUGGAGGCAGAAAGACC >hsa-mir-378c MI0015825 (SEQ ID NO: 1024)GGAGGCCAUCACUGGACUUGGAGUCAGAAGAGUGGAGUCGGGUCAGACUUCAACUCUGACUUUGAAGGUGGUGAGUGCCUC >hsa-mir-378d-1 MI0016749 (SEQ ID NO: 1025)ACUGUUUCUGUCCUUGUUCUUGUUGUUAUUACUGGACUUGGAGUCAGAAACAGG >hsa-mir-378d-2 MI0003840 (SEQ ID NO: 1026)GAAUGGUUACAAGGAGAGAACACUGGACUUGGAGUCAGAAAACUUUCAUCCAAGUCAUUCCCUGCUCUAAGUCCCAUUUCUGUUCCAUGAGAUUGUUU >hsa-mir-378e MI0016750 (SEQ ID NO: 1027)CUGACUCCAGUGUCCAGGCCAGGGGCAGACAGUGGACAGAGAACAGUGCCCAAGACCACUGGACUUGGAGUCAGGACAU >hsa-mir-378f MI0016756 (SEQ ID NO: 1028)GUCAGGUCCUGGACUCCCAUAGUUUUCAGGCUGCUAAACAACAGAACGAGCACUGGACUUGGAGCCAGAAGUCUUGGG >hsa-mir-378g MI0016761 (SEQ ID NO: 1029)CACUGGGCUUGGAGUCAGAAGACCUGGCUCCAGCCCAGCUC >hsa-mir-378h MI0016808 (SEQ ID NO: 1030)ACAGGAACACUGGACUUGGUGUCAGAUGGGAUGAGCCCUGGCUCUGUUUCCUAGCAGCAAUCUGAUCUUGAGCUAGUCACUGG >hsa-mir-378i MI0016902 (SEQ ID NO: 1031)GGGAGCACUGGACUAGGAGUCAGAAGGUGGAGUUCUGGGUGCUGUUUUCCCACUCUUGGGCCCUGGGCAUGUUCUG >hsa-mir-379 MI0000787 (SEQ ID NO: 1032)AGAGAUGGUAGACUAUGGAACGUAGGCGUUAUGAUUUCUGACCUAUGUAACAUGGUCCACUAACUCU >hsa-mir-380 MI0000788 (SEQ ID NO: 1033)AAGAUGGUUGACCAUAGAACAUGCGCUAUCUCUGUGUCGUAUGUAAUAUGGUCCACAUCUU >hsa-mir-381 MI0000789 (SEQ ID NO: 1034)UACUUAAAGCGAGGUUGCCCUUUGUAUAUUCGGUUUAUUGACAUGGAAUAUACAAGGGCAAGCUCUCUGUGAGUA >hsa-mir-382 MI0000790 (SEQ ID NO: 1035)UACUUGAAGAGAAGUUGUUCGUGGUGGAUUCGCUUUACUUAUGACGAAUCAUUCACGGACAACACUUUUUUCAGUA >hsa-mir-383 MI0000791 (SEQ ID NO: 1036)CUCCUCAGAUCAGAAGGUGAUUGUGGCUUUGGGUGGAUAUUAAUCAGCCACAGCACUGCCUGGUCAGAAAGAG >hsa-mir-384 MI0001145 (SEQ ID NO: 1037)UGUUAAAUCAGGAAUUUUAAACAAUUCCUAGACAAUAUGUAUAAUGUUCAUAAGUCAUUCCUAGAAAUUGUUCAUAAUGCCUGUAACA >hsa-mir-409 MI0001735 (SEQ ID NO: 1038)UGGUACUCGGGGAGAGGUUACCCGAGCAACUUUGCAUCUGGACGACGAAUGUUGCUCGGUGAACCCCUUUUCGGUAUCA >hsa-mir-410 MI0002465 (SEQ ID NO: 1039)GGUACCUGAGAAGAGGUUGUCUGUGAUGAGUUCGCUUUUAUUAAUGACGAAUAUAACACAGAUGGCCUGUUUUCAGUACC >hsa-mir-411 MI0003675 (SEQ ID NO: 1040)UGGUACUUGGAGAGAUAGUAGACCGUAUAGCGUACGCUUUAUCUGUGACGUAUGUAACACGGUCCACUAACCCUCAGUAUCAAAUCCAUCCCCGAG >hsa-mir-412 MI0002464 (SEQ ID NO: 1041)CUGGGGUACGGGGAUGGAUGGUCGACCAGUUGGAAAGUAAUUGUUUCUAAUGUACUUCACCUGGUCCACUAGCCGUCCGUAUCCGCUGCAG >hsa-mir-421 MI0003685 (SEQ ID NO: 1042)GCACAUUGUAGGCCUCAUUAAAUGUUUGUUGAAUGAAAAAAUGAAUCAUCAACAGACAUUAAUUGGGCGCCUGCUCUGUGAUCUC >hsa-mir-422a MI0001444 (SEQ ID NO: 1043)GAGAGAAGCACUGGACUUAGGGUCAGAAGGCCUGAGUCUCUCUGCUGCAGAUGGGCUCUCUGUCCCUGAGCCAAGCUUUGUCCUCCCUGG >hsa-mir-423 MI0001445 (SEQ ID NO: 1044)AUAAAGGAAGUUAGGCUGAGGGGCAGAGAGCGAGACUUUUCUAUUUUCCAAAAGCUCGGUCUGAGGCCCCUCAGUCUUGCUUCCUAACCCGCGC >hsa-mir-424 MI0001446 (SEQ ID NO: 1045)CGAGGGGAUACAGCAGCAAUUCAUGUUUUGAAGUGUUCUAAAUGGUUCAAAACGUGAGGCGCUGCUAUACCCCCUCGUGGGGAAGGUAGAAGGUGGGG >hsa-mir-425 MI0001448 (SEQ ID NO: 1046)GAAAGCGCUUUGGAAUGACACGAUCACUCCCGUUGAGUGGGCACCCGAGAAGCCAUCGGGAAUGUCGUGUCCGCCCAGUGCUCUUUC >hsa-mir-429 MI0001641 (SEQ ID NO: 1047)CGCCGGCCGAUGGGCGUCUUACCAGACAUGGUUAGACCUGGCCCUCUGUCUAAUACUGUCUGGUAAAACCGUCCAUCCGCUGC >hsa-mir-431 MI0001721 (SEQ ID NO: 1048)UCCUGCUUGUCCUGCGAGGUGUCUUGCAGGCCGUCAUGCAGGCCACACUGACGGUAACGUUGCAGGUCGUCUUGCAGGGCUUCUCGCAAGACGACAUCCUCAUCACCAACGACG >hsa-mir-432 MI0003133 (SEQ ID NO: 1049)UGACUCCUCCAGGUCUUGGAGUAGGUCAUUGGGUGGAUCCUCUAUUUCCUUACGUGGGCCACUGGAUGGCUCCUCCAUGUCUUGGAGUAGAUCA >hsa-mir-433 MI0001723 (SEQ ID NO: 1050)CCGGGGAGAAGUACGGUGAGCCUGUCAUUAUUCAGAGAGGCUAGAUCCUCUGUGUUGAGAAGGAUCAUGAUGGGCUCCUCGGUGUUCUCCAGG >hsa-mir-448 MI0001637 (SEQ ID NO: 1051)GCCGGGAGGUUGAACAUCCUGCAUAGUGCUGCCAGGAAAUCCCUAUUUCAUAUAAGAGGGGGCUGGCUGGUUGCAUAUGUAGGAUGUCCCAUCUCCCAGCCCACUUCGUCA >hsa-mir-449a MI0001648 (SEQ ID NO: 1052)CUGUGUGUGAUGAGCUGGCAGUGUAUUGUUAGCUGGUUGAAUAUGUGAAUGGCAUCGGCUAACAUGCAACUGCUGUCUUAUUGCAUAUACA >hsa-mir-449b MI0003673 (SEQ ID NO: 1053)UGACCUGAAUCAGGUAGGCAGUGUAUUGUUAGCUGGCUGCUUGGGUCAAGUCAGCAGCCACAACUACCCUGCCACUUGCUUCUGGAUAAAUUCUUCU >hsa-mir-449c MI0003823 (SEQ ID NO: 1054)GCUGGGAUGUGUCAGGUAGGCAGUGUAUUGCUAGCGGCUGUUAAUGAUUUUAACAGUUGCUAGUUGCACUCCUCUCUGUUGCAUUCAGAAGC >hsa-mir-450a-1 MI0001652 (SEQ ID NO: 1055)AAACGAUACUAAACUGUUUUUGCGAUGUGUUCCUAAUAUGCACUAUAAAUAUAUUGGGAACAUUUUGCAUGUAUAGUUUUGUAUCAAUAUA >hsa-mir-450a-2 MI0003187 (SEQ ID NO: 1056)CCAAAGAAAGAUGCUAAACUAUUUUUGCGAUGUGUUCCUAAUAUGUAAUAUAAAUGUAUUGGGGACAUUUUGCAUUCAUAGUUUUGUAUCAAUAAUAUGG >hsa-mir-450b MI0005531 (SEQ ID NO: 1057)GCAGAAUUAUUUUUGCAAUAUGUUCCUGAAUAUGUAAUAUAAGUGUAUUGGGAUCAUUUUGCAUCCAUAGUUUUGUAU >hsa-mir-451a MI0001729 (SEQ ID NO: 1058)CUUGGGAAUGGCAAGGAAACCGUUACCAUUACUGAGUUUAGUAAUGGUAAUGGUUCUCUUGCUAUACCCAGA >hsa-mir-451b MI0017360 (SEQ ID NO: 1059)UGGGUAUAGCAAGAGAACCAUUACCAUUACUAAACUCAGUAAUGGUAACGGUUUCCUUGCCAUUCCCA >hsa-mir-452 MI0001733 (SEQ ID NO: 1060)GCUAAGCACUUACAACUGUUUGCAGAGGAAACUGAGACUUUGUAACUAUGUCUCAGUCUCAUCUGCAAAGAAGUAAGUGCUUUGC >hsa-mir-454 MI0003820 (SEQ ID NO: 1061)UCUGUUUAUCACCAGAUCCUAGAACCCUAUCAAUAUUGUCUCUGCUGUGUAAAUAGUUCUGAGUAGUGCAAUAUUGCUUAUAGGGUUUUGGUGUUUGGAAAGAACAAUGGGCAGG >hsa-mir-455 MI0003513 (SEQ ID NO: 1062)UCCCUGGCGUGAGGGUAUGUGCCUUUGGACUACAUCGUGGAAGCCAGCACCAUGCAGUCCAUGGGCAUAUACACUUGCCUCAAGGCCUAUGUCAUC >hsa-mir-466 MI0014157 (SEQ ID NO: 1063)GUGUGUGUAUAUGUGUGUUGCAUGUGUGUAUAUGUGUGUAUAUAUGUACACAUACACAUACACGCAACACACAUAUAUACAUGC >hsa-mir-483 MI0002467 (SEQ ID NO: 1064)GAGGGGGAAGACGGGAGGAAAGAAGGGAGUGGUUCCAUCACGCCUCCUCACUCCUCUCCUCCCGUCUUCUCCUCUC >hsa-mir-484 MI0002468 (SEQ ID NO: 1065)AGCCUCGUCAGGCUCAGUCCCCUCCCGAUAAACCCCUAAAUAGGGACUUUCCCGGGGGGUGACCCUGGCUUUUUUGGCG >hsa-mir-485 MI0002469 (SEQ ID NO: 1066)ACUUGGAGAGAGGCUGGCCGUGAUGAAUUCGAUUCAUCAAAGCGAGUCAUACACGGCUCUCCUCUCUUUUAGU >hsa-mir-486 MI0002470 (SEQ ID NO: 1067)GCAUCCUGUACUGAGCUGCCCCGAGGCCCUUCAUGCUGCCCAGCUCGGGGCAGCUCAGUACAGGAUAC >hsa-mir-487a MI0002471 (SEQ ID NO: 1068)GGUACUUGAAGAGUGGUUAUCCCUGCUGUGUUCGCUUAAUUUAUGACGAAUCAUACAGGGACAUCCAGUUUUUCAGUAUC >hsa-mir-487b MI0003530 (SEQ ID NO: 1069)UUGGUACUUGGAGAGUGGUUAUCCCUGUCCUGUUCGUUUUGCUCAUGUCGAAUCGUACAGGGUCAUCCACUUUUUCAGUAUCAA >hsa-mir-488 MI0003123 (SEQ ID NO: 1070)GAGAAUCAUCUCUCCCAGAUAAUGGCACUCUCAAACAAGUUUCCAAAUUGUUUGAAAGGCUAUUUCUUGGUCAGAUGACUCUC >hsa-mir-489 MI0003124 (SEQ ID NO: 1071)GUGGCAGCUUGGUGGUCGUAUGUGUGACGCCAUUUACUUGAACCUUUAGGAGUGACAUCACAUAUACGGCAGCUAAACUGCUAC >hsa-mir-490 MI0003125 (SEQ ID NO: 1072)UGGAGGCCUUGCUGGUUUGGAAAGUUCAUUGUUCGACACCAUGGAUCUCCAGGUGGGUCAAGUUUAGAGAUGCACCAACCUGGAGGACUCCAUGCUGUUGAGCUGUUCACAAGCAGCGGACACUUCCA >hsa-mir-491 MI0003126 (SEQ ID NO: 1073)UUGACUUAGCUGGGUAGUGGGGAACCCUUCCAUGAGGAGUAGAACACUCCUUAUGCAAGAUUCCCUUCUACCUGGCUGGGUUGG >hsa-mir-492 MI0003131 (SEQ ID NO: 1074)CAACUACAGCCACUACUACAGGACCAUCGAGGACCUGCGGGACAAGAUUCUUGGUGCCACCAUUGAGAACGCCAGGAUUGUCCUGCAGAUCAACAAUGCUCAACUGGCUGCAGAUG >hsa-mir-493 MI0003132 (SEQ ID NO: 1075)CUGGCCUCCAGGGCUUUGUACAUGGUAGGCUUUCAUUCAUUCGUUUGCACAUUCGGUGAAGGUCUACUGUGUGCCAGGCCCUGUGCCAG >hsa-mir-494 MI0003134 (SEQ ID NO: 1076)GAUACUCGAAGGAGAGGUUGUCCGUGUUGUCUUCUCUUUAUUUAUGAUGAAACAUACACGGGAAACCUCUUUUUUAGUAUC >hsa-mir-495 MI0003135 (SEQ ID NO: 1077)UGGUACCUGAAAAGAAGUUGCCCAUGUUAUUUUCGCUUUAUAUGUGACGAAACAAACAUGGUGCACUUCUUUUUCGGUAUCA >hsa-mir-496 MI0003136 (SEQ ID NO: 1078)CCCAAGUCAGGUACUCGAAUGGAGGUUGUCCAUGGUGUGUUCAUUUUAUUUAUGAUGAGUAUUACAUGGCCAAUCUCCUUUCGGUACUCAAUUCUUCUUGGG >hsa-mir-497 MI0003138 (SEQ ID NO: 1079)CCACCCCGGUCCUGCUCCCGCCCCAGCAGCACACUGUGGUUUGUACGGCACUGUGGCCACGUCCAAACCACACUGUGGUGUUAGAGCGAGGGUGGGGGAGGCACCGCCGAGG >hsa-mir-498 MI0003142 (SEQ ID NO: 1080)AACCCUCCUUGGGAAGUGAAGCUCAGGCUGUGAUUUCAAGCCAGGGGGCGUUUUUCUAUAACUGGAUGAAAAGCACCUCCAGAGCUUGAAGCUCACAGUUUGAGAGCAAUCGUCUAAGGAAGUU >hsa-mir-499a MI0003183 (SEQ ID NO: 1081)GCCCUGUCCCCUGUGCCUUGGGCGGGCGGCUGUUAAGACUUGCAGUGAUGUUUAACUCCUCUCCACGUGAACAUCACAGCAAGUCUGUGCUGCUUCCCGUCCCUACGCUGCCUGGGCAGGGU >hsa-mir-499b MI0017396 (SEQ ID NO: 1082)GGAAGCAGCACAGACUUGCUGUGAUGUUCACGUGGAGAGGAGUUAAACAUCACUGCAAGUCUUAACAGCCGCC >hsa-mir-500a MI0003184 (SEQ ID NO: 1083)GCUCCCCCUCUCUAAUCCUUGCUACCUGGGUGAGAGUGCUGUCUGAAUGCAAUGCACCUGGGCAAGGAUUCUGAGAGCGAGAGC >hsa-mir-500b MI0015903 (SEQ ID NO: 1084)CCCCCUCUCUAAUCCUUGCUACCUGGGUGAGAGUGCUUUCUGAAUGCAGUGCACCCAGGCAAGGAUUCUGCAAGGGGGA >hsa-mir-501 MI0003185 (SEQ ID NO: 1085)GCUCUUCCUCUCUAAUCCUUUGUCCCUGGGUGAGAGUGCUUUCUGAAUGCAAUGCACCCGGGCAAGGAUUCUGAGAGGGUGAGC >hsa-mir-502 MI0003186 (SEQ ID NO: 1086)UGCUCCCCCUCUCUAAUCCUUGCUAUCUGGGUGCUAGUGCUGGCUCAAUGCAAUGCACCUGGGCAAGGAUUCAGAGAGGGGGAGCU >hsa-mir-503 MI0003188 (SEQ ID NO: 1087)UGCCCUAGCAGCGGGAACAGUUCUGCAGUGAGCGAUCGGUGCUCUGGGGUAUUGUUUCCGCUGCCAGGGUA >hsa-mir-504 MI0003189 (SEQ ID NO: 1088)GCUGCUGUUGGGAGACCCUGGUCUGCACUCUAUCUGUAUUCUUACUGAAGGGAGUGCAGGGCAGGGUUUCCCAUACAGAGGGC >hsa-mir-505 MI0003190 (SEQ ID NO: 1089)GAUGCACCCAGUGGGGGAGCCAGGAAGUAUUGAUGUUUCUGCCAGUUUAGCGUCAACACUUGCUGGUUUCCUCUCUGGAGCAUC >hsa-mir-506 MI0003193 (SEQ ID NO: 1090)GCCACCACCAUCAGCCAUACUAUGUGUAGUGCCUUAUUCAGGAAGGUGUUACUUAAUAGAUUAAUAUUUGUAAGGCACCCUUCUGAGUAGAGUAAUGUGCAACAUGGACAACAUUUGUGGUGGC >hsa-mir-507 MI0003194 (SEQ ID NO: 1091)GUGCUGUGUGUAGUGCUUCACUUCAAGAAGUGCCAUGCAUGUGUCUAGAAAUAUGUUUUGCACCUUUUGGAGUGAAAUAAUGCACAACAGAUAC >hsa-mir-508 MI0003195 (SEQ ID NO: 1092)CCACCUUCAGCUGAGUGUAGUGCCCUACUCCAGAGGGCGUCACUCAUGUAAACUAAAACAUGAUUGUAGCCUUUUGGAGUAGAGUAAUACACAUCACGUAACGCAUAUUUGGUGG >hsa-mir-509-1 MI0003196 (SEQ ID NO: 1093)CAUGCUGUGUGUGGUACCCUACUGCAGACAGUGGCAAUCAUGUAUAAUUAAAAAUGAUUGGUACGUCUGUGGGUAGAGUACUGCAUGACACAUG >hsa-mir-509-2 MI0005530 (SEQ ID NO: 1094)CAUGCUGUGUGUGGUACCCUACUGCAGACAGUGGCAAUCAUGUAUAAUUAAAAAUGAUUGGUACGUCUGUGGGUAGAGUACUGCAUGACAC >hsa-mir-509-3 MI0005717 (SEQ ID NO: 1095)GUGGUACCCUACUGCAGACGUGGCAAUCAUGUAUAAUUAAAAAUGAUUGGUACGUCUGUGGGUAGAGUACUGCAU >hsa-mir-510 MI0003197 (SEQ ID NO: 1096)GUGGUGUCCUACUCAGGAGAGUGGCAAUCACAUGUAAUUAGGUGUGAUUGAAACCUCUAAGAGUGGAGUAACAC >hsa-mir-511-1 MI0003127 (SEQ ID NO: 1097)CAAUAGACACCCAUCGUGUCUUUUGCUCUGCAGUCAGUAAAUAUUUUUUUGUGAAUGUGUAGCAAAAGACAGAAUGGUGGUCCAUUG >hsa-mir-511-2 MI0003128 (SEQ ID NO: 1098)CAAUAGACACCCAUCGUGUCUUUUGCUCUGCAGUCAGUAAAUAUUUUUUUGUGAAUGUGUAGCAAAAGACAGAAUGGUGGUCCAUUG >hsa-mir-512-1 MI0003140 (SEQ ID NO: 1099)UCUCAGUCUGUGGCACUCAGCCUUGAGGGCACUUUCUGGUGCCAGAAUGAAAGUGCUGUCAUAGCUGAGGUCCAAUGACUGAGG >hsa-mir-512-2 MI0003141 (SEQ ID NO: 1100)GGUACUUCUCAGUCUGUGGCACUCAGCCUUGAGGGCACUUUCUGGUGCCAGAAUGAAAGUGCUGUCAUAGCUGAGGUCCAAUGACUGAGGCGAGCACC >hsa-mir-513a-1 MI0003191 (SEQ ID NO: 1101)GGGAUGCCACAUUCAGCCAUUCAGCGUACAGUGCCUUUCACAGGGAGGUGUCAUUUAUGUGAACUAAAAUAUAAAUUUCACCUUUCUGAGAAGGGUAAUGUACAGCAUGCACUGCAUAUGUGGUGUCCC >hsa-mir-513a-2 MI0003192 (SEQ ID NO: 1102)GGAUGCCACAUUCAGCCAUUCAGUGUGCAGUGCCUUUCACAGGGAGGUGUCAUUUAUGUGAACUAAAAUAUAAAUUUCACCUUUCUGAGAAGGGUAAUGUACAGCAUGCACUGCAUAUGUGGUGUCC >hsa-mir-513b MI0006648 (SEQ ID NO: 1103)GUGUACAGUGCCUUUCACAAGGAGGUGUCAUUUAUGUGAACUAAAAUAUAAAUGUCACCUUUUUGAGAGGAGUAAUGUACAGCA >hsa-mir-513c MI0006649 (SEQ ID NO: 1104)GCGUACAGUGCCUUUCUCAAGGAGGUGUCGUUUAUGUGAACUAAAAUAUAAAUUUCACCUUUCUGAGAAGAGUAAUGUACAGCA >hsa-mir-514a-1 MI0003198 (SEQ ID NO: 1105)AACAUGUUGUCUGUGGUACCCUACUCUGGAGAGUGACAAUCAUGUAUAAUUAAAUUUGAUUGACACUUCUGUGAGUAGAGUAACGCAUGACACGUACG >hsa-mir-514a-2 MI0003199 (SEQ ID NO: 1106)GUUGUCUGUGGUACCCUACUCUGGAGAGUGACAAUCAUGUAUAACUAAAUUUGAUUGACACUUCUGUGAGUAGAGUAACGCAUGACAC >hsa-mir-514a-3 MI0003200 (SEQ ID NO: 1107)GUUGUCUGUGGUACCCUACUCUGGAGAGUGACAAUCAUGUAUAACUAAAUUUGAUUGACACUUCUGUGAGUAGAGUAACGCAUGACAC >hsa-mir-514b MI0014251 (SEQ ID NO: 1108)CAUGUGGUACUCUUCUCAAGAGGGAGGCAAUCAUGUGUAAUUAGAUAUGAUUGACACCUCUGUGAGUGGAGUAACACAUG >hsa-mir-515-1 MI0003144 (SEQ ID NO: 1109)UCUCAUGCAGUCAUUCUCCAAAAGAAAGCACUUUCUGUUGUCUGAAAGCAGAGUGCCUUCUUUUGGAGCGUUACUGUUUGAGA >hsa-mir-515-2 MI0003147 (SEQ ID NO: 1110)UCUCAUGCAGUCAUUCUCCAAAAGAAAGCACUUUCUGUUGUCUGAAAGCAGAGUGCCUUCUUUUGGAGCGUUACUGUUUGAGA >hsa-mir-516a-1 MI0003180 (SEQ ID NO: 1111)UCUCAGGCUGUGACCUUCUCGAGGAAAGAAGCACUUUCUGUUGUCUGAAAGAAAAGAAAGUGCUUCCUUUCAGAGGGUUACGGUUUGAGA >hsa-mir-516a-2 MI0003181 (SEQ ID NO: 1112)UCUCAGGUUGUGACCUUCUCGAGGAAAGAAGCACUUUCUGUUGUCUGAAAGAAAAGAAAGUGCUUCCUUUCAGAGGGUUACGGUUUGAGA >hsa-mir-516b-1 MI0003172 (SEQ ID NO: 1113)UCUCAGGCUGUGACCAUCUGGAGGUAAGAAGCACUUUCUGUUUUGUGAAAGAAAAGAAAGUGCUUCCUUUCAGAGGGUUACUCUUUGAGA >hsa-mir-516b-2 MI0003167 (SEQ ID NO: 1114)UCUCAUGAUGUGACCAUCUGGAGGUAAGAAGCACUUUGUGUUUUGUGAAAGAAAGUGCUUCCUUUCAGAGGGUUACUCUUUGAGA >hsa-mir-517a MI0003161 (SEQ ID NO: 1115)UCUCAGGCAGUGACCCUCUAGAUGGAAGCACUGUCUGUUGUAUAAAAGAAAAGAUCGUGCAUCCCUUUAGAGUGUUACUGUUUGAGA >hsa-mir-517b MI0003165 (SEQ ID NO: 1116)GUGACCCUCUAGAUGGAAGCACUGUCUGUUGUCUAAGAAAAGAUCGUGCAUCCCUUUAGAGUGUUAC >hsa-mir-517c MI0003174 (SEQ ID NO: 1117)GAAGAUCUCAGGCAGUGACCCUCUAGAUGGAAGCACUGUCUGUUGUCUAAGAAAAGAUCGUGCAUCCUUUUAGAGUGUUACUGUUUGAGAAAAUC >hsa-mir-518a-1 MI0003170 (SEQ ID NO: 1118)UCUCAAGCUGUGACUGCAAAGGGAAGCCCUUUCUGUUGUCUGAAAGAAGAGAAAGCGCUUCCCUUUGCUGGAUUACGGUUUGAGA >hsa-mir-518a-2 MI0003173 (SEQ ID NO: 1119)UCUCAAGCUGUGGGUCUGCAAAGGGAAGCCCUUUCUGUUGUCUAAAAGAAGAGAAAGCGCUUCCCUUUGCUGGAUUACGGUUUGAGA >hsa-mir-518b MI0003156 (SEQ ID NO: 1120)UCAUGCUGUGGCCCUCCAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAACAAAGCGCUCCCCUUUAGAGGUUUACGGUUUGA >hsa-mir-518c MI0003159 (SEQ ID NO: 1121)GCGAGAAGAUCUCAUGCUGUGACUCUCUGGAGGGAAGCACUUUCUGUUGUCUGAAAGAAAACAAAGCGCUUCUCUUUAGAGUGUUACGGUUUGAGAAAAGC >hsa-mir-518d MI0003171 (SEQ ID NO: 1122)UCCCAUGCUGUGACCCUCUAGAGGGAAGCACUUUCUGUUGUCUGAAAGAAACCAAAGCGCUUCCCUUUGGAGCGUUACGGUUUGAGA >hsa-mir-518e MI0003169 (SEQ ID NO: 1123)UCUCAGGCUGUGACCCUCUAGAGGGAAGCGCUUUCUGUUGGCUAAAAGAAAAGAAAGCGCUUCCCUUCAGAGUGUUAACGCUUUGAGA >hsa-mir-518f MI0003154 (SEQ ID NO: 1124)UCUCAUGCUGUGACCCUCUAGAGGGAAGCACUUUCUCUUGUCUAAAAGAAAAGAAAGCGCUUCUCUUUAGAGGAUUACUCUUUGAGA >hsa-mir-519a-1 MI0003178 (SEQ ID NO: 1125)CUCAGGCUGUGACACUCUAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAGGAAAGUGCAUCCUUUUAGAGUGUUACUGUUUGAG >hsa-mir-519a-2 MI0003182 (SEQ ID NO: 1126)UCUCAGGCUGUGUCCCUCUACAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAGGAAAGUGCAUCCUUUUAGAGUGUUACUGUUUGAGA >hsa-mir-519b MI0003151 (SEQ ID NO: 1127)CAUGCUGUGACCCUCUAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAAGAAAGUGCAUCCUUUUAGAGGUUUACUGUUUG >hsa-mir-519c MI0003148 (SEQ ID NO: 1128)UCUCAGCCUGUGACCCUCUAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAAGAAAGUGCAUCUUUUUAGAGGAUUACAGUUUGAGA >hsa-mir-519d MI0003162 (SEQ ID NO: 1129)UCCCAUGCUGUGACCCUCCAAAGGGAAGCGCUUUCUGUUUGUUUUCUCUUAAACAAAGUGCCUCCCUUUAGAGUGUUACCGUUUGGGA >hsa-mir-519e MI0003145 (SEQ ID NO: 1130)UCUCAUGCAGUCAUUCUCCAAAAGGGAGCACUUUCUGUUUGAAAGAAAACAAAGUGCCUCCUUUUAGAGUGUUACUGUUUGAGA >hsa-mir-520a MI0003149 (SEQ ID NO: 1131)CUCAGGCUGUGACCCUCCAGAGGGAAGUACUUUCUGUUGUCUGAGAGAAAAGAAAGUGCUUCCCUUUGGACUGUUUCGGUUUGAG >hsa-mir-520b MI0003155 (SEQ ID NO: 1132)CCCUCUACAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAAGAAAGUGCUUCCUUUUAGAGGG >hsa-mir-520c MI0003158 (SEQ ID NO: 1133)UCUCAGGCUGUCGUCCUCUAGAGGGAAGCACUUUCUGUUGUCUGAAAGAAAAGAAAGUGCUUCCUUUUAGAGGGUUACCGUUUGAGA >hsa-mir-520d MI0003164 (SEQ ID NO: 1134)UCUCAAGCUGUGAGUCUACAAAGGGAAGCCCUUUCUGUUGUCUAAAAGAAAAGAAAGUGCUUCUCUUUGGUGGGUUACGGUUUGAGA >hsa-mir-520e MI0003143 (SEQ ID NO: 1135)UCUCCUGCUGUGACCCUCAAGAUGGAAGCAGUUUCUGUUGUCUGAAAGGAAAGAAAGUGCUUCCUUUUUGAGGGUUACUGUUUGAGA >hsa-mir-520f MI0003146 (SEQ ID NO: 1136)UCUCAGGCUGUGACCCUCUAAAGGGAAGCGCUUUCUGUGGUCAGAAAGAAAAGCAAGUGCUUCCUUUUAGAGGGUUACCGUUUGGGA >hsa-mir-520g MI0003166 (SEQ ID NO: 1137)UCCCAUGCUGUGACCCUCUAGAGGAAGCACUUUCUGUUUGUUGUCUGAGAAAAAACAAAGUGCUUCCCUUUAGAGUGUUACCGUUUGGGA >hsa-mir-520h MI0003175 (SEQ ID NO: 1138)UCCCAUGCUGUGACCCUCUAGAGGAAGCACUUUCUGUUUGUUGUCUGAGAAAAAACAAAGUGCUUCCCUUUAGAGUUACUGUUUGGGA >hsa-mir-521-1 MI0003176 (SEQ ID NO: 1139)UCUCAGGCUGUGACCCUCCAAAGGGAAGAACUUUCUGUUGUCUAAAAGAAAAGAACGCACUUCCCUUUAGAGUGUUACCGUGUGAGA >hsa-mir-521-2 MI0003163 (SEQ ID NO: 1140)UCUCGGGCUGUGACUCUCCAAAGGGAAGAAUUUUCUCUUGUCUAAAAGAAAAGAACGCACUUCCCUUUAGAGUGUUACCGUGUGAGA >hsa-mir-522 MI0003177 (SEQ ID NO: 1141)UCUCAGGCUGUGUCCCUCUAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAAGAAAAUGGUUCCCUUUAGAGUGUUACGCUUUGAGA >hsa-mir-523 MI0003153 (SEQ ID NO: 1142)UCUCAUGCUGUGACCCUCUAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAAGAACGCGCUUCCCUAUAGAGGGUUACCCUUUGAGA >hsa-mir-524 MI0003160 (SEQ ID NO: 1143)UCUCAUGCUGUGACCCUACAAAGGGAAGCACUUUCUCUUGUCCAAAGGAAAAGAAGGCGCUUCCCUUUGGAGUGUUACGGUUUGAGA >hsa-mir-525 MI0003152 (SEQ ID NO: 1144)CUCAAGCUGUGACUCUCCAGAGGGAUGCACUUUCUCUUAUGUGAAAAAAAAGAAGGCGCUUCCCUUUAGAGCGUUACGGUUUGGG >hsa-mir-526a-1 MI0003157 (SEQ ID NO: 1145)CUCAGGCUGUGACCCUCUAGAGGGAAGCACUUUCUGUUGCUUGAAAGAAGAGAAAGCGCUUCCUUUUAGAGGAUUACUCUUUGAG >hsa-mir-526a-2 MI0003168 (SEQ ID NO: 1146)GUGACCCUCUAGAGGGAAGCACUUUCUGUUGAAAGAAAAGAACAUGCAUCCUUUCAGAGGGUUAC >hsa-mir-526b MI0003150 (SEQ ID NO: 1147)UCAGGCUGUGACCCUCUUGAGGGAAGCACUUUCUGUUGUCUGAAAGAAGAGAAAGUGCUUCCUUUUAGAGGCUUACUGUCUGA >hsa-mir-527 MI0003179 (SEQ ID NO: 1148)UCUCAAGCUGUGACUGCAAAGGGAAGCCCUUUCUGUUGUCUAAAAGAAAAGAAAGUGCUUCCCUUUGGUGAAUUACGGUUUGAGA >hsa-mir-532 MI0003205 (SEQ ID NO: 1149)CGACUUGCUUUCUCUCCUCCAUGCCUUGAGUGUAGGACCGUUGGCAUCUUAAUUACCCUCCCACACCCAAGGCUUGCAAAAAAGCGAGCCU >hsa-mir-539 MI0003514 (SEQ ID NO: 1150)AUACUUGAGGAGAAAUUAUCCUUGGUGUGUUCGCUUUAUUUAUGAUGAAUCAUACAAGGACAAUUUCUUUUUGAGUAU >hsa-mir-541 MI0005539 (SEQ ID NO: 1151)ACGUCAGGGAAAGGAUUCUGCUGUCGGUCCCACUCCAAAGUUCACAGAAUGGGUGGUGGGCACAGAAUCUGGACUCUGCUUGUG >hsa-mir-542 MI0003686 (SEQ ID NO: 1152)CAGAUCUCAGACAUCUCGGGGAUCAUCAUGUCACGAGAUACCAGUGUGCACUUGUGACAGAUUGAUAACUGAAAGGUCUGGGAGCCACUCAUCUUCA >hsa-mir-543 MI0005565 (SEQ ID NO: 1153)UACUUAAUGAGAAGUUGCCCGUGUUUUUUUCGCUUUAUUUGUGACGAAACAUUCGCGGUGCACUUCUUUUUCAGUAUC >hsa-mir-544a MI0003515 (SEQ ID NO: 1154)AUUUUCAUCACCUAGGGAUCUUGUUAAAAAGCAGAUUCUGAUUCAGGGACCAAGAUUCUGCAUUUUUAGCAAGUUCUCAAGUGAUGCUAAU >hsa-mir-544b MI0014159 (SEQ ID NO: 1155)GGAAUUUUGUUAAAAUGCAGAAUCCAUUUCUGUAGCUCUGAGACUAGACCUGAGGUUGUGCAUUUCUAACAAAGUGCC >hsa-mir-545 MI0003516 (SEQ ID NO: 1156)CCCAGCCUGGCACAUUAGUAGGCCUCAGUAAAUGUUUAUUAGAUGAAUAAAUGAAUGACUCAUCAGCAAACAUUUAUUGUGUGCCUGCUAAAGUGAGCUCCACAGG >hsa-mir-548a-1 MI0003593 (SEQ ID NO: 1157)UGCAGGGAGGUAUUAAGUUGGUGCAAAAGUAAUUGUGAUUUUUGCCAUUAAAAGUAACGACAAAACUGGCAAUUACUUUUGCACCAAACCUGGUAUU >hsa-mir-548a-2 MI0003598 (SEQ ID NO: 1158)UGUGAUGUGUAUUAGGUUUGUGCAAAAGUAAUUGGGGUUUUUUGCCGUUAAAAGUAAUGGCAAAACUGGCAAUUACUUUUGCACCAAACUAAUAUAA >hsa-mir-548a-3 MI0003612 (SEQ ID NO: 1159)CCUAGAAUGUUAUUAGGUCGGUGCAAAAGUAAUUGCGAGUUUUACCAUUACUUUCAAUGGCAAAACUGGCAAUUACUUUUGCACCAACGUAAUACUU >hsa-mir-548aa-1 MI0016689 (SEQ ID NO: 1160)CUUUAUUAGUCUGGUGCAAAAGAAACUGUGGUUUUUGCCAUUACUUUUACAGGCAAAAACCACAAUUACUUUUGCACCAACCUAAUAUAACUUGUUU >hsa-mir-548aa-2 MI0016690 (SEQ ID NO: 1161)UUUUAUUAGGUUGGUGCAAAAGAAACUGUGGUUUUUGCCAUUACUUUCAAUGGCAAAAACCACAAUUACUUUUGCACCAACCUAAAUCUUCCCUCUC >hsa-mir-548ab MI0016752 (SEQ ID NO: 1162)AUGUUGGUGCAAAAGUAAUUGUGGAUUUUGCUAUUACUUGUAUUUAUUUGUAAUGCAAAACCCGCAAUUAGUUUUGCACCAACC >hsa-mir-548ac MI0016762 (SEQ ID NO: 1163)GUAUUAGGUUGGUGCAAAAGUUAUUGUGGUUUUUGCUAUUUUUUUUUAAUGGCAAAAACCGGCAAUUACUUUUGCACUAACCUAGUAG >hsa-mir-548ad MI0016770 (SEQ ID NO: 1164)CUGUUAGGUUGGUGCAAAAGUAAUUGUGGUUUUUGAAAGUAACUUGGCGAAAACGACAAUGACUUUUGCACCAAUCUAAUAC >hsa-mir-548ae-1 MI0016779 (SEQ ID NO: 1165)GCAGUUUUUGCCAUUAAGUUGCGGUUUUUGCCAUUAUAAUGGCAAAAACUGCAAUUACUUUCACACCUGC >hsa-mir-548ae-2 MI0016780 (SEQ ID NO: 1166)UGUGCAAAAGUAAUUGUGGUUUUUGUCAUUUAAAAGUAAUGGCAAAAACUGCAAUUACUUUCACACC >hsa-mir-548ag-1 MI0016793 (SEQ ID NO: 1167)GUGCAAAGGUAAUUGUGGUUUCUGCUUUUAAAGGUAAUGGCAAAUAUUACAUUUACUUUUGCACCA >hsa-mir-548ag-2 MI0016794 (SEQ ID NO: 1168)UGCAAAGGUAAUUGUGGUUUCUGCCAUUGAAAGUAAAGGCAAGAACCUCAAUUACCUUUGCAGC >hsa-mir-548ah MI0016796 (SEQ ID NO: 1169)AGGUUGGUGCAAAAGUGAUUGCAGUGUUUGCCAAUAAAAGUAAUGACAAAAACUGCAGUUACUUUUGCACCAGCCC >hsa-mir-548ai MI0016813 (SEQ ID NO: 1170)GUAUUAGGUUGGUGCAAAGGUAAUUGCAGUUUUUCCCAUUUAAAAUAUGGAAAAAAAAAUCACAAUUACUUUUGCAUCAACCUAAUAA >hsa-mir-548aj-1 MI0016814 (SEQ ID NO: 1171)AUUGGUGUAAAAGUAAUUGCAGGUUAUGCCAUUAAAAGUAAUGGUAAAAACUGCAAUUACUUUUACACUAAC >hsa-mir-548aj-2 MI0016815 (SEQ ID NO: 1172)AAGGUAUUAGGUUGGUGCAAAAGUAAUUGCAGUUUUUGCUAUUACUUUUAAUGGUAAAAACUGCAAUUACUUUUACACCAACCUAAUAUUUA >hsa-mir-548ak MI0016840 (SEQ ID NO: 1173)GUGCAAAAGUAACUGCGGUUUUUGAGAAGUAAUUGAAAACCGCAAUUACUUUUGCAG >hsa-mir-548a1 MI0016851 (SEQ ID NO: 1174)GGUCGGUGCAAAAGUAAUUGCUGUUUUUGCCAUUAAAAAUAAUGGCAUUAAAAGUAAUGGCAAAAACGGCAAUGACUUUUGUACCAAUCUAAUAUCU >hsa-mir-548am MI0016904 (SEQ ID NO: 1175)AGUUGGUGCAAAAGUAAUUGCGGUUUUUGCCGUCGAAAAUAAUGGCAAAAACUGCAGUUACUUUUGUACCAAUG >hsa-mir-548an MI0016907 (SEQ ID NO: 1176)CAUUAGGUUGGUGCAAAAGGCAUUGUGGUUUUUGCCUAUAAAAGUAAUGGCAAAAACCGCAAUUCCUUUUGCACCAACCUAAU >hsa-mir-548ao MI0017871 (SEQ ID NO: 1177)AACUAUUCUUAGGUUGAUGCAGAAGUAACUACGGUUUUUGCAGUUGAAAGUAAUGGCAAAGACCGUGACUACUUUUGCAACAGCCUAAUAGUUUCU >hsa-mir-548ap MI0017875 (SEQ ID NO: 1178)ACCAAUUCCUAGGUUGGUGCAAAAGUAAUUGCGGUCUUUGUCAUUAAAACCAAUAACAAAAACCACAAUUACUUUUUACUGACCUAAAGAUUAAUU >hsa-mir-548aq MI0019130 (SEQ ID NO: 1179)GAAAGUAAUUGCUGUUUUUGCCAUUACUUUCAGUGGCAAAAACUGCAAUUACUUUUGC >hsa-mir-548ar MI0019131 (SEQ ID NO: 1180)AAAAGUAAUUGCAGUUUUUGCUGUUGAACGUAGUGGUAAAACUGCAGUUAUUUUUGC >hsa-mir-548as MI0019132 (SEQ ID NO: 1181)AAAAGUAAUUGCGGGUUUUGCCGUUGCUUUUAAUGGUAAAACCCACAAUUAUGUUUGU >hsa-mir-548at MI0019137 (SEQ ID NO: 1182)AAAAGUUAUUGCGGUUUUGGCUGCCAAAAGAAAUGGCCAAAACCGCAGUAACUUUUGU >hsa-mir-548au MI0019145 (SEQ ID NO: 1183)AAAAGUAAUUGCGGUUUUUGCUAUUGGUUUUAAUGGCAGUUACUUUUGCACCAG >hsa-mir-548av MI0019152 (SEQ ID NO: 1184)AAAAGUACUUGCGGAUUUGCCAUCACCUUUACCUUUAAUGGCAAAACUGCAGUUACUUUUGC >hsa-mir-548aw MI0019283 (SEQ ID NO: 1185)UAGGUCGGUGCAAAAGUCAUCACGGUUUUUACCAUUAAAACCGCGAUGACUUUUGCAUCAACCUA >hsa-mir-548ax MI0019286 (SEQ ID NO: 1186)GAUUGGUGCAGAAGUAAUUGCGGUUUUGCCAUGGAAAGUAAUGGCAAAAACCGUAAUUACUUUUGUACCAACC >hsa-mir-548b MI0003596 (SEQ ID NO: 1187)CAGACUAUAUAUUUAGGUUGGCGCAAAAGUAAUUGUGGUUUUGGCCUUUAUUUUCAAUGGCAAGAACCUCAGUUGCUUUUGUGCCAACCUAAUACUU >hsa-mir-548c MI0003630 (SEQ ID NO: 1188)CAUUGGCAUCUAUUAGGUUGGUGCAAAAGUAAUUGCGGUUUUUGCCAUUACUUUCAGUAGCAAAAAUCUCAAUUACUUUUGCACCAACUUAAUACUU >hsa-mir-548d-1 MI0003668 (SEQ ID NO: 1189)AAACAAGUUAUAUUAGGUUGGUGCAAAAGUAAUUGUGGUUUUUGCCUGUAAAAGUAAUGGCAAAAACCACAGUUUCUUUUGCACCAGACUAAUAAAG >hsa-mir-548d-2 MI0003671 (SEQ ID NO: 1190)GAGAGGGAAGAUUUAGGUUGGUGCAAAAGUAAUUGUGGUUUUUGCCAUUGAAAGUAAUGGCAAAAACCACAGUUUCUUUUGCACCAACCUAAUAAAA >hsa-mir-548e MI0006344 (SEQ ID NO: 1191)UUAUUAGGUUGGUACAAAAGCAAUCGCGGUUUUUGCUAUUACUUUUAAAGGCAAAAACUGAGACUACUUUUGCACCAACCUGAUAGAA >hsa-mir-548f-1 MI0006374 (SEQ ID NO: 1192)AUUAGGUUGGUGCAAAAGUAAUCACAGUUUUUGACAUUACUUUCAAAGACAAAAACUGUAAUUACUUUUGGACCAACCUAAUAG >hsa-mir-548f-2 MI0006375 (SEQ ID NO: 1193)UAAUAACUAUUAGGUUGGUGCGAACAUAAUUGCAGUUUUUAUCAUUACUUUUAAUGGCAAAAACUGUAAUUACUUUUGCACCAACCUAAUAUUUUAGU >hsa-mir-548f-3 MI0006376 (SEQ ID NO: 1194)AUUAGGUUGGUGCAAACCUAAUUGCAAUUUUUGCAGUUUUUUUAAGUAAUUGCAAAAACUGUAAUUACUUUUGCACCAACCUAAUAC >hsa-mir-548f-4 MI0006377 (SEQ ID NO: 1195)GAGUUCUAACGUAUUAGGUUGGUGCAAAAGUAAUAGUGGUUUUUGCCAUUAAAAGUAAUGACAAAAACUGUAAUUACUUUUGGAACAAUAUUAAUAGAAUUUCAG >hsa-mir-548f-5 MI0006378 (SEQ ID NO: 1196)UAUUAGGUUGCUGCAAAAGUAAUCAUGUUUUUUUCCAUUGUAAGUAAUGGGAAAAACUGUAAUUACUUUUGUACCAACCUAAUAGC >hsa-mir-548g MI0006395 (SEQ ID NO: 1197)AGUUAUUAGAUUAGUGCAAAAGUAAUUGCAGUUUUUGCAUUACGUUCUAUGGCAAAACUGUAAUUACUUUUGUACCAACAUAAUACUUC >hsa-mir-548h-1 MI0006411 (SEQ ID NO: 1198)UCUGUCCAUUAGGUGGGUGCAAAAGUAAUCGCGGUUUUUGUCAUUACUUUUAAUGGUAAAAACUGGAAUUACUUUUGCACUGACCUAAUAUUAAGCCAGAUA >hsa-mir-548h-2 MI0006412 (SEQ ID NO: 1199)GUAUUAGGUUGGUGCAAAAGUAAUCGCGGUUUUUGUCAUUACUUUCAAUGGCAAACACCACAAUUACUUUUGCACCAACCUAAUAUAA >hsa-mir-548h-3 MI0006413 (SEQ ID NO: 1200)UCUGAUUCUGCAUGUAUUAGGUUGGUGCAAAAGUAAUCGCGGUUUUUGUCAUUGAAAGUAAUAGCAAAAACUGCAAUUACUUUUGCACCAACCUAAAAGUAGUCACUGUCUUCAGAUA >hsa-mir-548h-4 MI0006414 (SEQ ID NO: 1201)GCUAUUAGGUUGGUGCAAAAGUAAUCGCGGUUUUUGUCAUUACUUUAAUUACUUUACGUUUCAUUAAUGACAAAAACCGCAAUUACUUUUGCACCAACCUAAUACUUGCUA >hsa-mir-548h-5 MI0016751 (SEQ ID NO: 1202)ACAAAAGUAAUCGCGGUUUUUGUCAUUACUUUUAACUGUAAAAACCACGGUUGCUUUUGC >hsa-mir-548i-1 MI0006421 (SEQ ID NO: 1203)CAGAUGGCUCUGAAGUUUGCACCCUAUUAGGUUGGUGCAAAAGUAAUUGCGGAUUUUGCCAUUAAAAGUAAUGGCAAAAAUAGCAAUUAUUUUUGUACCAGCCUAGUAUCUUUUCUCCUUCUACCAAACUUUGUCCCUGAGCCAUCUCA >hsa-mir-548i-2 MI0006422 (SEQ ID NO: 1204)UAGAUGGCUCCGAAGUUUGCAUCCUAUUAGUUUGGUGCAAAAGUAAUUGCGGAUUUUGCCAUUAAAAGUAAUGGCAAAAAUAGCAAUUAUUUUUGUACCAGCCUAGUAUCUUUUCUCCUUCUAACAAAGUUCGUCCCUGAUCCAUCUCA >hsa-mir-548i-3 MI0006423 (SEQ ID NO: 1205)CAGAUGGCUCCGAAGUUUACAUCCUAUUAGGUUUGUGCAAAAGUAAUUGCGGAUUUUGCCAUUAAAAGUAAUGGCAAAAAUAGCAAUUAUUUUUGUACCAGCCUAGUAUCUUUUCUCCUUCUACCAAACUUUGUCCCUGAGCCAUCUCA >hsa-mir-548i-4 MI0006424 (SEQ ID NO: 1206)AGGUUGGUGCAAAAGUAAUUGCGGAUUUUGCCAUACUUUUAACGGCAAAAACCACAAAUAUUAUUGCACCAACCUAU >hsa-mir-548j MI0006345 (SEQ ID NO: 1207)GGGCAGCCAGUGAAUAGUUAGCUGGUGCAAAAGUAAUUGCGGUCUUUGGUAUUACUUUCAGUGGCAAAAACUGCAUUACUUUUGCACCAGCCUACUAGAACGCUGAGUUCAG >hsa-mir-548k MI0006354 (SEQ ID NO: 1208)CUUUUCUCAAGUAUUGCUGUUAGGUUGGUGCAAAAGUACUUGCGGAUUUUGCUUUACUUUUAAUGGCAAAAACCGCAAUUAUUUUUGCUUCAACCUAAUAUGAUGCAAAAUUGGCU >hsa-mir-5481 MI0006361 (SEQ ID NO: 1209)UAUUAGGUUGGUGCAAAAGUAUUUGCGGGUUUUGUCGUAGAAAGUAAUGGCAAAAACUGCAGUUACUUGUGCACCAACCAAAUGCU >hsa-mir-548m MI0006400 (SEQ ID NO: 1210)AUAUUAGGUUGGUGCAAAGGUAUUUGUGGUUUUUGUCAUUAAAGUAAUGCAAAAGCCACAAAUACCUUUGCACCAACCUAAUAUUA >hsa-mir-548n MI0006399 (SEQ ID NO: 1211)AGGUUGGUGCAAAAGUAAUUGUGGAUUUUGUCGUUAAAAAUAGCAAAACCCGCAAUUACUUUUGCACCAACCUAA >hsa-mir-548o MI0006402 (SEQ ID NO: 1212)UGGUGAAAAUGUGUUGAUUGUAAUGGUUCCUAUUCUGAUCAAUAAACAUGGUUUGAGCCUAGUUACAAUGAUCUAAAAUUCACGGUCCAAAACUGCAGUUACUUUUGCACCAAC >hsa-mir-548o-2 MI0016746 (SEQ ID NO: 1213)UGGUGCAAAAGUAAUUGCGGUUUUUGCCAUUAAAAGUAAUGCGGCCAAAACUGCAGUUACUUUUGCACCC >hsa-mir-548p MI0006420 (SEQ ID NO: 1214)AUUAGGUUGGUAUAAAAUUAAUUGCAGUUUUUGUCAUUACUUUCAAUAGCAAAAACUGCAGUUACUUUUGCACCAAUGUAAUAC >hsa-mir-548q MI0010637 (SEQ ID NO: 1215)AUAUUAGGCUGGUGCAAAAGUAAUGGCGGUUUUUGCCAUUACUUUUCAUUUUUACCAUUAAAAGUAAUGGCAAAAAGCAUGAUUACUUUUUCACCAACCU >hsa-mir-548s MI0014141 (SEQ ID NO: 1216)UUGCUGCAAAAAUAAUUGCAGUUUUUGCCAUUAUUUUUAAUAAUUAUAAUAAUGGCCAAAACUGCAGUUAUUUUUGCACCAA >hsa-mir-548t MI0014164 (SEQ ID NO: 1217)AGGGUGGUGCAAAAGUGAUCGUGGUUUUUGCAAUUUUUUAAUGACAAAAACCACAAUUACUUUUGCACCAACCU >hsa-mir-548u MI0014168 (SEQ ID NO: 1218)AUUAGGAUGGUGCAAAAGUAAUGUGGUUUUUUUCUUUACUUUUAAUGGCAAAGACUGCAAUUACUUUUGCGCCAACCUAAU >hsa-mir-548v MI0014174 (SEQ ID NO: 1219)AAUACUAGGUUUGAGCAAAAGUAAUUGCGGUUUUGCCAUCAUGCCAAAAGCUACAGUUACUUUUGCACCAGCCUAAUAUU >hsa-mir-548w MI0014222 (SEQ ID NO: 1220)GGUUGGUGCAAAAGUAACUGCGGUUUUUGCCUUUCAACAUAAUGGCAAAACCCACAAUUACUUUUGCACCAAUC >hsa-mir-548x MI0014244 (SEQ ID NO: 1221)AGGUUAGUGCAAAAGUAAUUGCAGUUUUUGCGUUACUUUCAAUCGUAAAAACUGCAAUUACUUUCACACCAAUCU >hsa-mir-548x-2 MI0016833 (SEQ ID NO: 1222)AUGCCAAAUAUUAGGUUGGCACAAAAGUAAUUGUGGCUUUUGCCAUUAAAAGUAAUGGUAAAAACUGCAAUUACUUUCGUGCCAACCUAAUAUUUGUGUG >hsa-mir-548y MI0016595 (SEQ ID NO: 1223)GCCUAAACUAUUAGGUUGGUGCAAAAGUAAUCACUGUUUUUGCCAUUACUCUCAGUGGCAAAAACCGUGAUUACUUUUGCACCAACCUAGUAACACCUUCACUGUGGGGG >hsa-mir-548z MI0016688 (SEQ ID NO: 1224)AAGUAUUAAGUUGGUGCAAAAGUAAUUGAGAUUUUUGCUACUGAAAGUAAUGGCAAAAACCGCAAUUACUUUUGCACCAACCUAAUAGAUGCCAAUG >hsa-mir-549 MI0003679 (SEQ ID NO: 1225)AGACAUGCAACUCAAGAAUAUAUUGAGAGCUCAUCCAUAGUUGUCACUGUCUCAAAUCAGUGACAACUAUGGAUGAGCUCUUAAUAUAUCCCAGGC >hsa-mir-550a-1 MI0003600 (SEQ ID NO: 1226)UGAUGCUUUGCUGGCUGGUGCAGUGCCUGAGGGAGUAAGAGCCCUGUUGUUGUAAGAUAGUGUCUUACUCCCUCAGGCACAUCUCCAACAAGUCUCU >hsa-mir-550a-2 MI0003601 (SEQ ID NO: 1227)UGAUGCUUUGCUGGCUGGUGCAGUGCCUGAGGGAGUAAGAGCCCUGUUGUUGUCAGAUAGUGUCUUACUCCCUCAGGCACAUCUCCAGCGAGUCUCU >hsa-mir-550a-3 MI0003762 (SEQ ID NO: 1228)GAUGCUUUGCUGGCUGGUGCAGUGCCUGAGGGAGUAAGAGUCCUGUUGUUGUAAGAUAGUGUCUUACUCCCUCAGGCACAUCUCCAACAAGUCUC >hsa-mir-550b-1 MI0016686 (SEQ ID NO: 1229)AGAGACUUGUUGGAGAUGUGCCUGAGGGAGUAAGACACUAUCUUACAACAACAGGGCUCUUACUCCCUCAGGCACUGCACCAGCCAGCAAAGCAUCA >hsa-mir-550b-2 MI0016687 (SEQ ID NO: 1230)AGAGACUCGCUGGAGAUGUGCCUGAGGGAGUAAGACACUAUCUGACAACAACAGGGCUCUUACUCCCUCAGGCACUGCACCAGCCAGCAAAGCAUCA >hsa-mir-551a MI0003556 (SEQ ID NO: 1231)GGGGACUGCCGGGUGACCCUGGAAAUCCAGAGUGGGUGGGGCCAGUCUGACCGUUUCUAGGCGACCCACUCUUGGUUUCCAGGGUUGCCCUGGAAA >hsa-mir-551b MI0003575 (SEQ ID NO: 1232)AGAUGUGCUCUCCUGGCCCAUGAAAUCAAGCGUGGGUGAGACCUGGUGCAGAACGGGAAGGCGACCCAUACUUGGUUUCAGAGGCUGUGAGAAUAA >hsa-mir-552 MI0003557 (SEQ ID NO: 1233)AACCAUUCAAAUAUACCACAGUUUGUUUAACCUUUUGCCUGUUGGUUGAAGAUGCCUUUCAACAGGUGACUGGUUAGACAAACUGUGGUAUAUACA >hsa-mir-553 MI0003558 (SEQ ID NO: 1234)CUUCAAUUUUAUUUUAAAACGGUGAGAUUUUGUUUUGUCUGAGAAAAUCUCGCUGUUUUAGACUGAGG >hsa-mir-554 MI0003559 (SEQ ID NO: 1235)ACCUGAGUAACCUUUGCUAGUCCUGACUCAGCCAGUACUGGUCUUAGACUGGUGAUGGGUCAGGGUUCAUAUUUUGGCAUCUCUCUCUGGGCAUCU >hsa-mir-555 MI0003561 (SEQ ID NO: 1236)GGAGUGAACUCAGAUGUGGAGCACUACCUUUGUGAGCAGUGUGACCCAAGGCCUGUGGACAGGGUAAGCUGAACCUCUGAUAAAACUCUGAUCUAU >hsa-mir-556 MI0003562 (SEQ ID NO: 1237)GAUAGUAAUAAGAAAGAUGAGCUCAUUGUAAUAUGAGCUUCAUUUAUACAUUUCAUAUUACCAUUAGCUCAUCUUUUUUAUUACUACCUUCAACA >hsa-mir-557 MI0003563 (SEQ ID NO: 1238)AGAAUGGGCAAAUGAACAGUAAAUUUGGAGGCCUGGGGCCCUCCCUGCUGCUGGAGAAGUGUUUGCACGGGUGGGCCUUGUCUUUGAAAGGAGGUGGA >hsa-mir-558 MI0003564 (SEQ ID NO: 1239)GUGUGUGUGUGUGUGUGUGGUUAUUUUGGUAUAGUAGCUCUAGACUCUAUUAUAGUUUCCUGAGCUGCUGUACCAAAAUACCACAAACGGGCUG >hsa-mir-559 MI0003565 (SEQ ID NO: 1240)GCUCCAGUAACAUCUUAAAGUAAAUAUGCACCAAAAUUACUUUUGGUAAAUACAGUUUUGGUGCAUAUUUACUUUAGGAUGUUACUGGAGCUCCCA >hsa-mir-561 MI0003567 (SEQ ID NO: 1241)CUUCAUCCACCAGUCCUCCAGGAACAUCAAGGAUCUUAAACUUUGCCAGAGCUACAAAGGCAAAGUUUAAGAUCCUUGAAGUUCCUGGGGGAACCAU >hsa-mir-562 MI0003568 (SEQ ID NO: 1242)AGUGAAAUUGCUAGGUCAUAUGGUCAGUCUACUUUUAGAGUAAUUGUGAAACUGUUUUUCAAAGUAGCUGUACCAUUUGCACUCCCUGUGGCAAU >hsa-mir-563 MI0003569 (SEQ ID NO: 1243)AGCAAAGAAGUGUGUUGCCCUCUAGGAAAUGUGUGUUGCUCUGAUGUAAUUAGGUUGACAUACGUUUCCCUGGUAGCCA >hsa-mir-564 MI0003570 (SEQ ID NO: 1244)CGGGCAGCGGGUGCCAGGCACGGUGUCAGCAGGCAACAUGGCCGAGAGGCCGGGGCCUCCGGGCGGCGCCGUGUCCGCGACCGCGUACCCUGAC >hsa-mir-566 MI0003572 (SEQ ID NO: 1245)GCUAGGCGUGGUGGCGGGCGCCUGUGAUCCCAACUACUCAGGAGGCUGGGGCAGCAGAAUCGCUUGAACCCGGGAGGCGAAGGUUGCAGUGAGC >hsa-mir-567 MI0003573 (SEQ ID NO: 1246)GGAUUCUUAUAGGACAGUAUGUUCUUCCAGGACAGAACAUUCUUUGCUAUUUUGUACUGGAAGAACAUGCAAAACUAAAAAAAAAAAAAGUUAUUGCU >hsa-mir-568 MI0003574 (SEQ ID NO: 1247)GAUAUACACUAUAUUAUGUAUAAAUGUAUACACACUUCCUAUAUGUAUCCACAUAUAUAUAGUGUAUAUAUUAUACAUGUAUAGGUGUGUAUAUG >hsa-mir-569 MI0003576 (SEQ ID NO: 1248)GGUAUUGUUAGAUUAAUUUUGUGGGACAUUAACAACAGCAUCAGAAGCAACAUCAGCUUUAGUUAAUGAAUCCUGGAAAGUUAAGUGACUUUAUUU >hsa-mir-570 MI0003577 (SEQ ID NO: 1249)CUAGAUAAGUUAUUAGGUGGGUGCAAAGGUAAUUGCAGUUUUUCCCAUUAUUUUAAUUGCGAAAACAGCAAUUACCUUUGCACCAACCUGAUGGAGU >hsa-mir-571 MI0003578 (SEQ ID NO: 1250)CCUCAGUAAGACCAAGCUCAGUGUGCCAUUUCCUUGUCUGUAGCCAUGUCUAUGGGCUCUUGAGUUGGCCAUCUGAGUGAGGGCCUGCUUAUUCUA >hsa-mir-572 MI0003579 (SEQ ID NO: 1251)GUCGAGGCCGUGGCCCGGAAGUGGUCGGGGCCGCUGCGGGCGGAAGGGCGCCUGUGCUUCGUCCGCUCGGCGGUGGCCCAGCCAGGCCCGCGGGA >hsa-mir-573 MI0003580 (SEQ ID NO: 1252)UUUAGCGGUUUCUCCCUGAAGUGAUGUGUAACUGAUCAGGAUCUACUCAUGUCGUCUUUGGUAAAGUUAUGUCGCUUGUCAGGGUGAGGAGAGUUUUUG >hsa-mir-574 MI0003581 (SEQ ID NO: 1253)GGGACCUGCGUGGGUGCGGGCGUGUGAGUGUGUGUGUGUGAGUGUGUGUCGCUCCGGGUCCACGCUCAUGCACACACCCACACGCCCACACUCAGG >hsa-mir-575 MI0003582 (SEQ ID NO: 1254)AAUUCAGCCCUGCCACUGGCUUAUGUCAUGACCUUGGGCUACUCAGGCUGUCUGCACAAUGAGCCAGUUGGACAGGAGCAGUGCCACUCAACUC >hsa-mir-576 MI0003583 (SEQ ID NO: 1255)UACAAUCCAACGAGGAUUCUAAUUUCUCCACGUCUUUGGUAAUAAGGUUUGGCAAAGAUGUGGAAAAAUUGGAAUCCUCAUUCGAUUGGUUAUAACCA >hsa-mir-577 MI0003584 (SEQ ID NO: 1256)UGGGGGAGUGAAGAGUAGAUAAAAUAUUGGUACCUGAUGAAUCUGAGGCCAGGUUUCAAUACUUUAUCUGCUCUUCAUUUCCCCAUAUCUACUUAC >hsa-mir-578 MI0003585 (SEQ ID NO: 1257)AGAUAAAUCUAUAGACAAAAUACAAUCCCGGACAACAAGAAGCUCCUAUAGCUCCUGUAGCUUCUUGUGCUCUAGGAUUGUAUUUUGUUUAUAUAU >hsa-mir-579 MI0003586 (SEQ ID NO: 1258)CAUAUUAGGUUAAUGCAAAAGUAAUCGCGGUUUGUGCCAGAUGACGAUUUGAAUUAAUAAAUUCAUUUGGUAUAAACCGCGAUUAUUUUUGCAUCAAC >hsa-mir-580 MI0003587 (SEQ ID NO: 1259)AUAAAAUUUCCAAUUGGAACCUAAUGAUUCAUCAGACUCAGAUAUUUAAGUUAACAGUAUUUGAGAAUGAUGAAUCAUUAGGUUCCGGUCAGAAAUU >hsa-mir-581 MI0003588 (SEQ ID NO: 1260)GUUAUGUGAAGGUAUUCUUGUGUUCUCUAGAUCAGUGCUUUUAGAAAAUUUGUGUGAUCUAAAGAACACAAAGAAUACCUACACAGAACCACCUGC >hsa-mir-582 MI0003589 (SEQ ID NO: 1261)AUCUGUGCUCUUUGAUUACAGUUGUUCAACCAGUUACUAAUCUAACUAAUUGUAACUGGUUGAACAACUGAACCCAAAGGGUGCAAAGUAGAAACAUU >hsa-mir-583 MI0003590 (SEQ ID NO: 1262)AACUCACACAUUAACCAAAGAGGAAGGUCCCAUUACUGCAGGGAUCUUAGCAGUACUGGGACCUACCUCUUUGGU >hsa-mir-584 MI0003591 (SEQ ID NO: 1263)UAGGGUGACCAGCCAUUAUGGUUUGCCUGGGACUGAGGAAUUUGCUGGGAUAUGUCAGUUCCAGGCCAACCAGGCUGGUUGGUCUCCCUGAAGCAAC >hsa-mir-585 MI0003592 (SEQ ID NO: 1264)UGGGGUGUCUGUGCUAUGGCAGCCCUAGCACACAGAUACGCCCAGAGAAAGCCUGAACGUUGGGCGUAUCUGUAUGCUAGGGCUGCUGUAACAA >hsa-mir-586 MI0003594 (SEQ ID NO: 1265)AUGGGGUAAAACCAUUAUGCAUUGUAUUUUUAGGUCCCAAUACAUGUGGGCCCUAAAAAUACAAUGCAUAAUGGUUUUUCACUCUUUAUCUUCUUAU >hsa-mir-587 MI0003595 (SEQ ID NO: 1266)CUCCUAUGCACCCUCUUUCCAUAGGUGAUGAGUCACAGGGCUCAGGGAAUGUGUCUGCACCUGUGACUCAUCACCAGUGGAAAGCCCAUCCCAUAU >hsa-mir-588 MI0003597 (SEQ ID NO: 1267)AGCUUAGGUACCAAUUUGGCCACAAUGGGUUAGAACACUAUUCCAUUGUGUUCUUACCCACCAUGGCCAAAAUUGGGCCUAAG >hsa-mir-589 MI0003599 (SEQ ID NO: 1268)UCCAGCCUGUGCCCAGCAGCCCCUGAGAACCACGUCUGCUCUGAGCUGGGUACUGCCUGUUCAGAACAAAUGCCGGUUCCCAGACGCUGCCAGCUGGCC >hsa-mir-590 MI0003602 (SEQ ID NO: 1269)UAGCCAGUCAGAAAUGAGCUUAUUCAUAAAAGUGCAGUAUGGUGAAGUCAAUCUGUAAUUUUAUGUAUAAGCUAGUCUCUGAUUGAAACAUGCAGCA >hsa-mir-591 MI0003603 (SEQ ID NO: 1270)UCUUAUCAAUGAGGUAGACCAUGGGUUCUCAUUGUAAUAGUGUAGAAUGUUGGUUAACUGUGGACUCCCUGGCUCUGUCUCAAAUCUACUGAUUC >hsa-mir-592 MI0003604 (SEQ ID NO: 1271)UAUUAUGCCAUGACAUUGUGUCAAUAUGCGAUGAUGUGUUGUGAUGGCACAGCGUCAUCACGUGGUGACGCAACAUCAUGACGUAAGACGUCACAAC >hsa-mir-593 MI0003605 (SEQ ID NO: 1272)CCCCCAGAAUCUGUCAGGCACCAGCCAGGCAUUGCUCAGCCCGUUUCCCUCUGGGGGAGCAAGGAGUGGUGCUGGGUUUGUCUCUGCUGGGGUUUCUCCU >hsa-mir-595 MI0003607 (SEQ ID NO: 1273)ACGGAAGCCUGCACGCAUUUAACACCAGCACGCUCAAUGUAGUCUUGUAAGGAACAGGUUGAAGUGUGCCGUGGUGUGUCUGGAGGAAGCGCCUGU >hsa-mir-596 MI0003608 (SEQ ID NO: 1274)AGCACGGCCUCUCCGAAGCCUGCCCGGCUCCUCGGGAACCUGCCUCCCGCAUGGCAGCUGCUGCCCUUCGGAGGCCG >hsa-mir-597 MI0003609 (SEQ ID NO: 1275)UACUUACUCUACGUGUGUGUCACUCGAUGACCACUGUGAAGACAGUAAAAUGUACAGUGGUUCUCUUGUGGCUCAAGCGUAAUGUAGAGUACUGGUC >hsa-mir-598 MI0003610 (SEQ ID NO: 1276)GCUUGAUGAUGCUGCUGAUGCUGGCGGUGAUCCCGAUGGUGUGAGCUGGAAAUGGGGUGCUACGUCAUCGUUGUCAUCGUCAUCAUCAUCAUCCGAG >hsa-mir-599 MI0003611 (SEQ ID NO: 1277)AAAGACAUGCUGUCCACAGUGUGUUUGAUAAGCUGACAUGGGACAGGGAUUCUUUUCACUGUUGUGUCAGUUUAUCAAACCCAUACUUGGAUGAC >hsa-mir-600 MI0003613 (SEQ ID NO: 1278)AAGUCACGUGCUGUGGCUCCAGCUUCAUAGGAAGGCUCUUGUCUGUCAGGCAGUGGAGUUACUUACAGACAAGAGCCUUGCUCAGGCCAGCCCUGCCC >hsa-mir-601 MI0003614 (SEQ ID NO: 1279)UGCAUGAGUUCGUCUUGGUCUAGGAUUGUUGGAGGAGUCAGAAAAACUACCCCAGGGAUCCUGAAGUCCUUUGGGUGGA >hsa-mir-602 MI0003615 (SEQ ID NO: 1280)UUCUCACCCCCGCCUGACACGGGCGACAGCUGCGGCCCGCUGUGUUCACUCGGGCCGAGUGCGUCUCCUGUCAGGCAAGGGAGAGCAGAGCCCCCCUG >hsa-mir-603 MI0003616 (SEQ ID NO: 1281)GAUUGAUGCUGUUGGUUUGGUGCAAAAGUAAUUGCAGUGCUUCCCAUUUAAAAGUAAUGGCACACACUGCAAUUACUUUUGCUCCAACUUAAUACUU >hsa-mir-604 MI0003617 (SEQ ID NO: 1282)AGAGCAUCGUGCUUGACCUUCCACGCUCUCGUGUCCACUAGCAGGCAGGUUUUCUGACACAGGCUGCGGAAUUCAGGACAGUGCAUCAUGGAGA >hsa-mir-605 MI0003618 (SEQ ID NO: 1283)GCCCUAGCUUGGUUCUAAAUCCCAUGGUGCCUUCUCCUUGGGAAAAACAGAGAAGGCACUAUGAGAUUUAGAAUCAAGUUAGG >hsa-mir-606 MI0003619 (SEQ ID NO: 1284)UGUAUCCUUGGUUUUUAGUAGUUUUACUAUGAUGAGGUGUGCCAUCCACCCCAUCAUAGUAAACUACUGAAAAUCAAAGAUACAAGUGCCUGACCA >hsa-mir-607 MI0003620 (SEQ ID NO: 1285)UUGCCUAAAGUCACACAGGUUAUAGAUCUGGAUUGGAACCCAGGGAGCCAGACUGCCUGGGUUCAAAUCCAGAUCUAUAACUUGUGUGACUUUGGG >hsa-mir-608 MI0003621 (SEQ ID NO: 1286)GGGCCAAGGUGGGCCAGGGGUGGUGUUGGGACAGCUCCGUUUAAAAAGGCAUCUCCAAGAGCUUCCAUCAAAGGCUGCCUCUUGGUGCAGCACAGGUAGA >hsa-mir-609 MI0003622 (SEQ ID NO: 1287)UGCUCGGCUGUUCCUAGGGUGUUUCUCUCAUCUCUGGUCUAUAAUGGGUUAAAUAGUAGAGAUGAGGGCAACACCCUAGGAACAGCAGAGGAACC >hsa-mir-610 MI0003623 (SEQ ID NO: 1288)UCUAUUUGUCUUAGGUGAGCUAAAUGUGUGCUGGGACACAUUUGAGCCAAAUGUCCCAGCACACAUUUAGCUCACAUAAGAAAAAUGGACUCUAGU >hsa-mir-611 MI0003624 (SEQ ID NO: 1289)AAAAUGGUGAGAGCGUUGAGGGGAGUUCCAGACGGAGAUGCGAGGACCCCUCGGGGUCUGACCCACA >hsa-mir-612 MI0003625 (SEQ ID NO: 1290)UCCCAUCUGGACCCUGCUGGGCAGGGCUUCUGAGCUCCUUAGCACUAGCAGGAGGGGCUCCAGGGGCCCUCCCUCCAUGGCAGCCAGGACAGGACUCUCA >hsa-mir-613 MI0003626 (SEQ ID NO: 1291)GGUGAGUGCGUUUCCAAGUGUGAAGGGACCCUUCCUGUAGUGUCUUAUAUACAAUACAGUAGGAAUGUUCCUUCUUUGCCACUCAUACACCUUUA >hsa-mir-614 MI0003627 (SEQ ID NO: 1292)UCUAAGAAACGCAGUGGUCUCUGAAGCCUGCAGGGGCAGGCCAGCCCUGCACUGAACGCCUGUUCUUGCCAGGUGGCAGAAGGUUGCUGC >hsa-mir-615 MI0003628 (SEQ ID NO: 1293)CUCGGGAGGGGCGGGAGGGGGGUCCCCGGUGCUCGGAUCUCGAGGGUGCUUAUUGUUCGGUCCGAGCCUGGGUCUCCCUCUUCCCCCCAACCCCCC >hsa-mir-616 MI0003629 (SEQ ID NO: 1294)UUAGGUAAUUCCUCCACUCAAAACCCUUCAGUGACUUCCAUGACAUGAAAUAGGAAGUCAUUGGAGGGUUUGAGCAGAGGAAUGACCUGUUUUAAAA >hsa-mir-617 MI0003631 (SEQ ID NO: 1295)CAUCAUAAGGAGCCUAGACUUCCCAUUUGAAGGUGGCCAUUUCCUACCACCUUCAAAUGGUAAGUCCAGGCUCCUUCUGAUUCAAUAAAUGAGGAGC >hsa-mir-618 MI0003632 (SEQ ID NO: 1296)CUCUUGUUCACAGCCAAACUCUACUUGUCCUUCUGAGUGUAAUUACGUACAUGCAGUAGCUCAGGAGACAAGCAGGUUUACCCUGUGGAUGAGUCUGA >hsa-mir-619 MI0003633 (SEQ ID NO: 1297)CGCCCACCUCAGCCUCCCAAAAUGCUGGGAUUACAGGCAUGAGCCACUGCGGUCGACCAUGACCUGGACAUGUUUGUGCCCAGUACUGUCAGUUUGCAG >hsa-mir-620 MI0003634 (SEQ ID NO: 1298)AUAUAUAUCUAUAUCUAGCUCCGUAUAUAUAUAUAUAUAUAUAUAGAUAUCUCCAUAUAUAUGGAGAUAGAUAUAGAAAUAAAACAAGCAAAGAA >hsa-mir-621 MI0003635 (SEQ ID NO: 1299)UAGAUUGAGGAAGGGGCUGAGUGGUAGGCGGUGCUGCUGUGCUCUGAUGAAGACCCAUGUGGCUAGCAACAGCGCUUACCUUUUGUCUCUGGGUCC >hsa-mir-622 MI0003636 (SEQ ID NO: 1300)AGAGAAGCUGGACAAGUACUGGUCUCAGCAGAUUGAGGAGAGCACCACAGUGGUCAUCACACAGUCUGCUGAGGUUGGAGCUGCUGAGAUGACACU >hsa-mir-623 MI0003637 (SEQ ID NO: 1301)GUACACAGUAGAAGCAUCCCUUGCAGGGGCUGUUGGGUUGCAUCCUAAGCUGUGCUGGAGCUUCCCGAUGUACUCUGUAGAUGUCUUUGCACCUUCUG >hsa-mir-624 MI0003638 (SEQ ID NO: 1302)AAUGCUGUUUCAAGGUAGUACCAGUACCUUGUGUUCAGUGGAACCAAGGUAAACACAAGGUAUUGGUAUUACCUUGAGAUAGCAUUACACCUAAGUG >hsa-mir-625 MI0003639 (SEQ ID NO: 1303)AGGGUAGAGGGAUGAGGGGGAAAGUUCUAUAGUCCUGUAAUUAGAUCUCAGGACUAUAGAACUUUCCCCCUCAUCCCUCUGCCCU >hsa-mir-626 MI0003640 (SEQ ID NO: 1304)ACUGAUAUAUUUGUCUUAUUUGAGAGCUGAGGAGUAUUUUUAUGCAAUCUGAAUGAUCUCAGCUGUCUGAAAAUGUCUUCAAUUUUAAAGGCUU >hsa-mir-627 MI0003641 (SEQ ID NO: 1305)UACUUAUUACUGGUAGUGAGUCUCUAAGAAAAGAGGAGGUGGUUGUUUUCCUCCUCUUUUCUUUGAGACUCACUACCAAUAAUAAGAAAUACUACUA >hsa-mir-628 MI0003642 (SEQ ID NO: 1306)AUAGCUGUUGUGUCACUUCCUCAUGCUGACAUAUUUACUAGAGGGUAAAAUUAAUAACCUUCUAGUAAGAGUGGCAGUCGAAGGGAAGGGCUCAU >hsa-mir-629 MI0003643 (SEQ ID NO: 1307)UCCCUUUCCCAGGGGAGGGGCUGGGUUUACGUUGGGAGAACUUUUACGGUGAACCAGGAGGUUCUCCCAACGUAAGCCCAGCCCCUCCCCUCUGCCU >hsa-mir-630 MI0003644 (SEQ ID NO: 1308)AACUUAACAUCAUGCUACCUCUUUGUAUCAUAUUUUGUUAUUCUGGUCACAGAAUGACCUAGUAUUCUGUACCAGGGAAGGUAGUUCUUAACUAUAU >hsa-mir-631 MI0003645 (SEQ ID NO: 1309)GUGGGGAGCCUGGUUAGACCUGGCCCAGACCUCAGCUACACAAGCUGAUGGACUGAGUCAGGGGCCACACUCUCC >hsa-mir-632 MI0003647 (SEQ ID NO: 1310)CGCCUCCUACCGCAGUGCUUGACGGGAGGCGGAGCGGGGAACGAGGCCGUCGGCCAUUUUGUGUCUGCUUCCUGUGGGACGUGGUGGUAGCCGU >hsa-mir-633 MI0003648 (SEQ ID NO: 1311)AACCUCUCUUAGCCUCUGUUUCUUUAUUGCGGUAGAUACUAUUAACCUAAAAUGAGAAGGCUAAUAGUAUCUACCACAAUAAAAUUGUUGUGAGGAUA >hsa-mir-634 MI0003649 (SEQ ID NO: 1312)AAACCCACACCACUGCAUUUUGGCCAUCGAGGGUUGGGGCUUGGUGUCAUGCCCCAAGAUAACCAGCACCCCAACUUUGGACAGCAUGGAUUAGUCU >hsa-mir-635 MI0003650 (SEQ ID NO: 1313)CAGAGAGGAGCUGCCACUUGGGCACUGAAACAAUGUCCAUUAGGCUUUGUUAUGGAAACUUCUCCUGAUCAUUGUUUUGUGUCCAUUGAGCUUCCAAU >hsa-mir-636 MI0003651 (SEQ ID NO: 1314)UGGCGGCCUGGGCGGGAGCGCGCGGGCGGGGCCGGCCCCGCUGCCUGGAAUUAACCCCGCUGUGCUUGCUCGUCCCGCCCGCAGCCCUAGGCGGCGUCG >hsa-mir-637 MI0003652 (SEQ ID NO: 1315)UGGCUAAGGUGUUGGCUCGGGCUCCCCACUGCAGUUACCCUCCCCUCGGCGUUACUGAGCACUGGGGGCUUUCGGGCUCUGCGUCUGCACAGAUACUUC >hsa-mir-638 MI0003653 (SEQ ID NO: 1316)GUGAGCGGGCGCGGCAGGGAUCGCGGGCGGGUGGCGGCCUAGGGCGCGGAGGGCGGACCGGGAAUGGCGCGCCGUGCGCCGCCGGCGUAACUGCGGCGCU >hsa-mir-639 MI0003654 (SEQ ID NO: 1317)UGGCCGACGGGGCGCGCGCGGCCUGGAGGGGCGGGGCGGACGCAGAGCCGCGUUUAGUCUAUCGCUGCGGUUGCGAGCGCUGUAGGGAGCCUGUGCUG >hsa-mir-640 MI0003655 (SEQ ID NO: 1318)GUGACCCUGGGCAAGUUCCUGAAGAUCAGACACAUCAGAUCCCUUAUCUGUAAAAUGGGCAUGAUCCAGGAACCUGCCUCUACGGUUGCCUUGGGG >hsa-mir-641 MI0003656 (SEQ ID NO: 1319)UGGGUGAAAGGAAGGAAAGACAUAGGAUAGAGUCACCUCUGUCCUCUGUCCUCUACCUAUAGAGGUGACUGUCCUAUGUCUUUCCUUCCUCUUACCCCU >hsa-mir-642a MI0003657 (SEQ ID NO: 1320)AUCUGAGUUGGGAGGGUCCCUCUCCAAAUGUGUCUUGGGGUGGGGGAUCAAGACACAUUUGGAGAGGGAACCUCCCAACUCGGCCUCUGCCAUCAUU >hsa-mir-642b MI0016685 (SEQ ID NO: 1321)GAGUUGGGAGGUUCCCUCUCCAAAUGUGUCUUGAUCCCCCACCCCAAGACACAUUUGGAGAGGGACCCUCCCAACUC >hsa-mir-643 MI0003658 (SEQ ID NO: 1322)ACCAAGUGAUAUUCAUUGUCUACCUGAGCUAGAAUACAAGUAGUUGGCGUCUUCAGAGACACUUGUAUGCUAGCUCAGGUAGAUAUUGAAUGAAAAA >hsa-mir-644a MI0003659 (SEQ ID NO: 1323)UUUUUUUUUAGUAUUUUUCCAUCAGUGUUCAUAAGGAAUGUUGCUCUGUAGUUUUCUUAUAGUGUGGCUUUCUUAGAGCAAAGAUGGUUCCCUA >hsa-mir-644b MI0019134 (SEQ ID NO: 1324)UGGGCUAAGGGAGAUGAUUGGGUAGAAAGUAUUAUUCUAUUCAUUUGCCUCCCAGCCUACA >hsa-mir-645 MI0003660 (SEQ ID NO: 1325)CAGUUCCUAACAGGCCUCAGACCAGUACCGGUCUGUGGCCUGGGGGUUGAGGACCCCUGCUCUAGGCUGGUACUGCUGAUGCUUAAAAAGAGAG >hsa-mir-646 MI0003661 (SEQ ID NO: 1326)GAUCAGGAGUCUGCCAGUGGAGUCAGCACACCUGCUUUUCACCUGUGAUCCCAGGAGAGGAAGCAGCUGCCUCUGAGGCCUCAGGCUCAGUGGC >hsa-mir-647 MI0003662 (SEQ ID NO: 1327)AGGAAGUGUUGGCCUGUGGCUGCACUCACUUCCUUCAGCCCCAGGAAGCCUUGGUCGGGGGCAGGAGGGAGGGUCAGGCAGGGCUGGGGGCCUGAC >hsa-mir-648 MI0003663 (SEQ ID NO: 1328)AUCACAGACACCUCCAAGUGUGCAGGGCACUGGUGGGGGCCGGGGCAGGCCCAGCGAAAGUGCAGGACCUGGCACUUAGUCGGAAGUGAGGGUG >hsa-mir-649 MI0003664 (SEQ ID NO: 1329)GGCCUAGCCAAAUACUGUAUUUUUGAUCGACAUUUGGUUGAAAAAUAUCUAUGUAUUAGUAAACCUGUGUUGUUCAAGAGUCCACUGUGUUUUGCUG >hsa-mir-650 MI0003665 (SEQ ID NO: 1330)CAGUGCUGGGGUCUCAGGAGGCAGCGCUCUCAGGACGUCACCACCAUGGCCUGGGCUCUGCUCCUCCUCACCCUCCUCACUCAGGGCACAGGUGAU >hsa-mir-651 MI0003666 (SEQ ID NO: 1331)AAUCUAUCACUGCUUUUUAGGAUAAGCUUGACUUUUGUUCAAAUAAAAAUGCAAAAGGAAAGUGUAUCCUAAAAGGCAAUGACAGUUUAAUGUGUUU >hsa-mir-652 MI0003667 (SEQ ID NO: 1332)ACGAAUGGCUAUGCACUGCACAACCCUAGGAGAGGGUGCCAUUCACAUAGACUAUAAUUGAAUGGCGCCACUAGGGUUGUGCAGUGCACAACCUACAC >hsa-mir-653 MI0003674 (SEQ ID NO: 1333)UUCAUUCCUUCAGUGUUGAAACAAUCUCUACUGAACCAGCUUCAAACAAGUUCACUGGAGUUUGUUUCAAUAUUGCAAGAAUGAUAAGAUGGAAGC >hsa-mir-654 MI0003676 (SEQ ID NO: 1334)GGGUAAGUGGAAAGAUGGUGGGCCGCAGAACAUGUGCUGAGUUCGUGCCAUAUGUCUGCUGACCAUCACCUUUAGAAGCCC >hsa-mir-655 MI0003677 (SEQ ID NO: 1335)AACUAUGCAAGGAUAUUUGAGGAGAGGUUAUCCGUGUUAUGUUCGCUUCAUUCAUCAUGAAUAAUACAUGGUUAACCUCUUUUUGAAUAUCAGACUC >hsa-mir-656 MI0003678 (SEQ ID NO: 1336)CUGAAAUAGGUUGCCUGUGAGGUGUUCACUUUCUAUAUGAUGAAUAUUAUACAGUCAACCUCUUUCCGAUAUCGAAUC >hsa-mir-657 MI0003681 (SEQ ID NO: 1337)GUGUAGUAGAGCUAGGAGGAGAGGGUCCUGGAGAAGCGUGGACCGGUCCGGGUGGGUUCCGGCAGGUUCUCACCCUCUCUAGGCCCCAUUCUCCUCUG >hsa-mir-658 MI0003682 (SEQ ID NO: 1338)GCUCGGUUGCCGUGGUUGCGGGCCCUGCCCGCCCGCCAGCUCGCUGACAGCACGACUCAGGGCGGAGGGAAGUAGGUCCGUUGGUCGGUCGGGAACGAGG >hsa-mir-659 MI0003683 (SEQ ID NO: 1339)UACCGACCCUCGAUUUGGUUCAGGACCUUCCCUGAACCAAGGAAGAGUCACAGUCUCUUCCUUGGUUCAGGGAGGGUCCCCAACAAUGUCCUCAUGG >hsa-mir-660 MI0003684 (SEQ ID NO: 1340)CUGCUCCUUCUCCCAUACCCAUUGCAUAUCGGAGUUGUGAAUUCUCAAAACACCUCCUGUGUGCAUGGAUUACAGGAGGGUGAGCCUUGUCAUCGUG >hsa-mir-661 MI0003669 (SEQ ID NO: 1341)GGAGAGGCUGUGCUGUGGGGCAGGCGCAGGCCUGAGCCCUGGUUUCGGGCUGCCUGGGUCUCUGGCCUGCGCGUGACUUUGGGGUGGCU >hsa-mir-662 MI0003670 (SEQ ID NO: 1342)GCUGUUGAGGCUGCGCAGCCAGGCCCUGACGGUGGGGUGGCUGCGGGCCUUCUGAAGGUCUCCCACGUUGUGGCCCAGCAGCGCAGUCACGUUGC >hsa-mir-663a MI0003672 (SEQ ID NO: 1343)CCUUCCGGCGUCCCAGGCGGGGCGCCGCGGGACCGCCCUCGUGUCUGUGGCGGUGGGAUCCCGCGGCCGUGUUUUCCUGGUGGCCCGGCCAUG >hsa-mir-663b MI0006336 (SEQ ID NO: 1344)GGUGCCGAGGGCCGUCCGGCAUCCUAGGCGGGUCGCUGCGGUACCUCCCUCCUGUCUGUGGCGGUGGGAUCCCGUGGCCGUGUUUUCCUGGUGGCCCGGCCGUGCCUGAGGUUUC >hsa-mir-664 MI0006442 (SEQ ID NO: 1345)GAACAUUGAAACUGGCUAGGGAAAAUGAUUGGAUAGAAACUAUUAUUCUAUUCAUUUAUCCCCAGCCUACAAAAUGAAAAAA >hsa-mir-665 MI0005563 (SEQ ID NO: 1346)UCUCCUCGAGGGGUCUCUGCCUCUACCCAGGACUCUUUCAUGACCAGGAGGCUGAGGCCCCUCACAGGCGGC >hsa-mir-668 MI0003761 (SEQ ID NO: 1347)GGUAAGUGCGCCUCGGGUGAGCAUGCACUUAAUGUGGGUGUAUGUCACUCGGCUCGGCCCACUACC >hsa-mir-670 MI0003933 (SEQ ID NO: 1348)GUUUAGGGGUGGACCUGAUGUCCCUGAGUGUAUGUGGUGAACCUGAAUUUGCCUUGGGUUUCCUCAUAUUCAUUCAGGAGUGUCAGUUGCCCCUUCAC >hsa-mir-671 MI0003760 (SEQ ID NO: 1349)GCAGGUGAACUGGCAGGCCAGGAAGAGGAGGAAGCCCUGGAGGGGCUGGAGGUGAUGGAUGUUUUCCUCCGGUUCUCAGGGCUCCACCUCUUUCGGGCCGUAGAGCCAGGGCUGGUGC >hsa-mir-675 MI0005416 (SEQ ID NO: 1350)CCCAGGGUCUGGUGCGGAGAGGGCCCACAGUGGACUUGGUGACGCUGUAUGCCCUCACCGCUCAGCCCCUGGG >hsa-mir-676 MI0016436 (SEQ ID NO: 1351)GCAUGACUCUUCAACCUCAGGACUUGCAGAAUUAAUGGAAUGCUGUCCUAAGGUUGUUGAGUUGUGC >hsa-mir-708 MI0005543 (SEQ ID NO: 1352)AACUGCCCUCAAGGAGCUUACAAUCUAGCUGGGGGUAAAUGACUUGCACAUGAACACAACUAGACUGUGAGCUUCUAGAGGGCAGGGA >hsa-mir-711 MI0012488 (SEQ ID NO: 1353)ACUGACUUUGAGUCUCUCCUCAGGGUGCUGCAGGCAAAGCUGGGGACCCAGGGAGAGACGUAAGUGAGGGGAGAUG >hsa-mir-718 MI0012489 (SEQ ID NO: 1354)GGCCGCGGCGCGCAAGAUGGCGGCGGGCCCGGGCACCGCCCCUUCCGCCCCGCCGGGCGUCGCACGAGGC >hsa-mir-720 MI0006654 (SEQ ID NO: 1355)CCGGAUCUCACACGGUGGUGUUAAUAUCUCGCUGGGGCCUCCAAAAUGUUGUGCCCAGGGGUGUUAGAGAAAACACCACACUUUGAGAUGAAUUAAGAGUCCUUUAUUAG >hsa-mir-744 MI0005559 (SEQ ID NO: 1356)UUGGGCAAGGUGCGGGGCUAGGGCUAACAGCAGUCUUACUGAAGGUUUCCUGGAAACCACGCACAUGCUGUUGCCACUAACCUCAACCUUACUCGGUC >hsa-mir-758 MI0003757 (SEQ ID NO: 1357)GCCUGGAUACAUGAGAUGGUUGACCAGAGAGCACACGCUUUAUUUGUGCCGUUUGUGACCUGGUCCACUAACCCUCAGUAUCUAAUGC >hsa-mir-759 MI0004065 (SEQ ID NO: 1358)UAAUAAAUUAAAUGCCUAAACUGGCAGAGUGCAAACAAUUUUGACUCAGAUCUAAAUGUUUGCACUGGCUGUUUAAACAUUUAAUUUGUUA >hsa-mir-760 MI0005567 (SEQ ID NO: 1359)GGCGCGUCGCCCCCCUCAGUCCACCAGAGCCCGGAUACCUCAGAAAUUCGGCUCUGGGUCUGUGGGGAGCGAAAUGCAAC >hsa-mir-761 MI0003941 (SEQ ID NO: 1360)GGAGGAGCAGCAGGGUGAAACUGACACAGUUCUGGUGAGUUUCACUUUGCUGCUCCUCC >hsa-mir-762 MI0003892 (SEQ ID NO: 1361)GGCCCGGCUCCGGGUCUCGGCCCGUACAGUCCGGCCGGCCAUGCUGGCGGGGCUGGGGCCGGGGCCGAGCCCGCGGCGGGGCC >hsa-mir-764 MI0003944 (SEQ ID NO: 1362)AAUCUAGGAGGCAGGUGCUCACUUGUCCUCCUCCAUGCUUGGAAAAUGCAGGGAGGAGGCCAUAGUGGCAACUGUUACCAUGAUU >hsa-mir-765 MI0005116 (SEQ ID NO: 1363)UUUAGGCGCUGAUGAAAGUGGAGUUCAGUAGACAGCCCUUUUCAAGCCCUACGAGAAACUGGGGUUUCUGGAGGAGAAGGAAGGUGAUGAAGGAUCUGUUCUCGUGAGCCUGAA >hsa-mir-766 MI0003836 (SEQ ID NO: 1364)GCAUCCUCAGGACCUGGGCUUGGGUGGUAGGAGGAAUUGGUGCUGGUCUUUCAUUUUGGAUUUGACUCCAGCCCCACAGCCUCAGCCACCCCAGCCAAUUGUCAUAGGAGC >hsa-mir-767 MI0003763 (SEQ ID NO: 1365)GCUUUUAUAUUGUAGGUUUUUGCUCAUGCACCAUGGUUGUCUGAGCAUGCAGCAUGCUUGUCUGCUCAUACCCCAUGGUUUCUGAGCAGGAACCUUCAUUGUCUACUGC >hsa-mir-769 MI0003834 (SEQ ID NO: 1366)GCCUUGGUGCUGAUUCCUGGGCUCUGACCUGAGACCUCUGGGUUCUGAGCUGUGAUGUUGCUCUCGAGCUGGGAUCUCCGGGGUCUUGGUUCAGGGCCGGGGCCUCUGGGUUCCAAGC >hsa-mir-770 MI0005118 (SEQ ID NO: 1367)AGGAGCCACCUUCCGAGCCUCCAGUACCACGUGUCAGGGCCACAUGAGCUGGGCCUCGUGGGCCUGAUGUGGUGCUGGGGCCUCAGGGGUCUGCUCUU >hsa-mir-802 MI0003906 (SEQ ID NO: 1368)GUUCUGUUAUUUGCAGUCAGUAACAAAGAUUCAUCCUUGUGUCCAUCAUGCAACAAGGAGAAUCUUUGUCACUUAGUGUAAUUAAUAGCUGGAC >hsa-mir-873 MI0005564 (SEQ ID NO: 1369)GUGUGCAUUUGCAGGAACUUGUGAGUCUCCUAUUGAAAAUGAACAGGAGACUGAUGAGUUCCCGGGAACACCCACAA >hsa-mir-874 MI0005532 (SEQ ID NO: 1370)UUAGCCCUGCGGCCCCACGCACCAGGGUAAGAGAGACUCUCGCUUCCUGCCCUGGCCCGAGGGACCGACUGGCUGGGC >hsa-mir-875 MI0005541 (SEQ ID NO: 1371)UUAGUGGUACUAUACCUCAGUUUUAUCAGGUGUUCUUAAAAUCACCUGGAAACACUGAGGUUGUGUCUCACUGAAC >hsa-mir-876 MI0005542 (SEQ ID NO: 1372)UGAAGUGCUGUGGAUUUCUUUGUGAAUCACCAUAUCUAAGCUAAUGUGGUGGUGGUUUACAAAGUAAUUCAUAGUGCUUCA >hsa-mir-877 MI0005561 (SEQ ID NO: 1373)GUAGAGGAGAUGGCGCAGGGGACACGGGCAAAGACUUGGGGGUUCCUGGGACCCUCAGACGUGUGUCCUCUUCUCCCUCCUCCCAG >hsa-mir-885 MI0005560 (SEQ ID NO: 1374)CCGCACUCUCUCCAUUACACUACCCUGCCUCUUCUCCAUGAGAGGCAGCGGGGUGUAGUGGAUAGAGCACGGGU >hsa-mir-887 MI0005562 (SEQ ID NO: 1375)GUGCAGAUCCUUGGGAGCCCUGUUAGACUCUGGAUUUUACACUUGGAGUGAACGGGCGCCAUCCCGAGGCUUUGCACAG >hsa-mir-888 MI0005537 (SEQ ID NO: 1376)GGCAGUGCUCUACUCAAAAAGCUGUCAGUCACUUAGAUUACAUGUGACUGACACCUCUUUGGGUGAAGGAAGGCUCA >hsa-mir-889 MI0005540 (SEQ ID NO: 1377)GUGCUUAAAGAAUGGCUGUCCGUAGUAUGGUCUCUAUAUUUAUGAUGAUUAAUAUCGGACAACCAUUGUUUUAGUAUCC >hsa-mir-890 MI0005533 (SEQ ID NO: 1378)GGAAGUGCCCUACUUGGAAAGGCAUCAGUUGCUUAGAUUACAUGUAACUAUUCCCUUUCUGAGUAGAGUAAGUCUUA >hsa-mir-891a MI0005524 (SEQ ID NO: 1379)CCUUAAUCCUUGCAACGAACCUGAGCCACUGAUUCAGUAAAAUACUCAGUGGCACAUGUUUGUUGUGAGGGUCAAAAGA >hsa-mir-891b MI0005534 (SEQ ID NO: 1380)CCUUAAUCCUUGCAACUUACCUGAGUCAUUGAUUCAGUAAAACAUUCAAUGGCACAUGUUUGUUGUUAGGGUCAAAAGA >hsa-mir-892a MI0005528 (SEQ ID NO: 1381)GCAGUGCCUUACUCAGAAAGGUGCCAGUCACUUACACUACAUGUCACUGUGUCCUUUCUGCGUAGAGUAAGGCUC >hsa-mir-892b MI0005538 (SEQ ID NO: 1382)UGCAAUGCCCUACUCAGAAAGGUGCCAUUUAUGUAGAUUUUAUGUCACUGGCUCCUUUCUGGGUAGAGCAAGGCUCA >hsa-mir-920 MI0005712 (SEQ ID NO: 1383)GUAGUUGUUCUACAGAAGACCUGGAUGUGUAGGAGCUAAGACACACUCCAGGGGAGCUGUGGAAGCAGUAACACG >hsa-mir-921 MI0005713 (SEQ ID NO: 1384)ACUAGUGAGGGACAGAACCAGGAUUCAGACUCAGGUCCAUGGGCCUGGAUCACUGG >hsa-mir-922 MI0005714 (SEQ ID NO: 1385)AUGGCGUUUUCCCUCUCCCUGUCCUGGACUGGGGUCAGACUGUGCCCCGAGGAGAAGCAGCAGAGAAUAGGACUACGUCAU >hsa-mir-924 MI0005716 (SEQ ID NO: 1386)AAUAGAGUCUUGUGAUGUCUUGCUUAAGGGCCAUCCAACCUAGAGUCUACAAC >hsa-mir-933 MI0005755 (SEQ ID NO: 1387)ACUUGGGUCAGUUCAGAGGUCCUCGGGGCGCGCGUCGAGUCAGCCGUGUGCGCAGGGAGACCUCUCCCACCCACAGU >hsa-mir-934 MI0005756 (SEQ ID NO: 1388)AGAAAUAAGGCUUCUGUCUACUACUGGAGACACUGGUAGUAUAAAACCCAGAGUCUCCAGUAAUGGACGGGAGCCUUAUUUCU >hsa-mir-935 MI0005757 (SEQ ID NO: 1389)GGCGGGGGCGCGGGCGGCAGUGGCGGGAGCGGCCCCUCGGCCAUCCUCCGUCUGCCCAGUUACCGCUUCCGCUACCGCCGCCGCUCCCGCU >hsa-mir-936 MI0005758 (SEQ ID NO: 1390)UCAAGGCCACUGGGACAGUAGAGGGAGGAAUCGCAGAAAUCACUCCAGGAGCAACUGAGAGACCUUGCUUCUACUUUACCAGGUCCUGCUGGCCCAGA >hsa-mir-937 MI0005759 (SEQ ID NO: 1391)AGCACUGCCCCCGGUGAGUCAGGGUGGGGCUGGCCCCCUGCUUCGUGCCCAUCCGCGCUCUGACUCUCUGCCCACCUGCAGGAGCU >hsa-mir-938 MI0005760 (SEQ ID NO: 1392)GAAGGUGUACCAUGUGCCCUUAAAGGUGAACCCAGUGCACCUUCAUGAACCGUGGUACACCUUUAAGAACUUGGUAUGCCUUC >hsa-mir-939 MI0005761 (SEQ ID NO: 1393)UGUGGGCAGGGCCCUGGGGAGCUGAGGCUCUGGGGGUGGCCGGGGCUGACCCUGGGCCUCUGCUCCCCAGUGUCUGACCGCG >hsa-mir-940 MI0005762 (SEQ ID NO: 1394)GUGAGGUGUGGGCCCGGCCCCAGGAGCGGGGCCUGGGCAGCCCCGUGUGUUGAGGAAGGAAGGCAGGGCCCCCGCUCCCCGGGCCUGACCCCAC >hsa-mir-941-1 MI0005763 (SEQ ID NO: 1395)CACGGAAGAGGACACACCCGGCUGUGUGGACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGG >hsa-mir-941-2 MI0005764 (SEQ ID NO: 1396)CACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGG >hsa-mir-941-3 MI0005765 (SEQ ID NO: 1397)CACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCAUGG >hsa-mir-941-4 MI0005766 (SEQ ID NO: 1398)CAUGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGG >hsa-mir-942 MI0005767 (SEQ ID NO: 1399)AUUAGGAGAGUAUCUUCUCUGUUUUGGCCAUGUGUGUACUCACAGCCCCUCACACAUGGCCGAAACAGAGAAGUUACUUUCCUAAU >hsa-mir-943 MI0005768 (SEQ ID NO: 1400)GGGACGUUCUGAGCUCGGGGUGGGGGACGUUUGCCGGUCACUGCUGCUGGCGCCCUGACUGUUGCCGUCCUCCAGCCCCACUCAAAGGCAUCCC >hsa-mir-944 MI0005769 (SEQ ID NO: 1401)GUUCCAGACACAUCUCAUCUGAUAUACAAUAUUUUCUUAAAUUGUAUAAAGAGAAAUUAUUGUACAUCGGAUGAGCUGUGUCUGGGAU >hsa-mir-1178 MI0006271 (SEQ ID NO: 1402)GCGUUGGCUGGCAGAGGAAGGGAAGGGUCCAGGGUCAGCUGAGCAUGCCCUCAGGUUGCUCACUGUUCUUCCCUAGAAUGUCAGGUGAUGU >hsa-mir-1179 MI0006272 (SEQ ID NO: 1403)GGCUGGAAAGGAAGAAGCAUUCUUUCAUUGGUUGGUGUGUAUUGCCUUGUCAACCAAUAAGAGGAUGCCAUUUAUCCUUUUCUGACUAGCU >hsa-mir-1180 MI0006273 (SEQ ID NO: 1404)GCUGCUGGACCCACCCGGCCGGGAAUAGUGCUCCUGGUUGUUUCCGGCUCGCGUGGGUGUGUCGGCGGC >hsa-mir-1181 MI0006274 (SEQ ID NO: 1405)UCCACUGCUGCCGCCGUCGCCGCCACCCGAGCCGGAGCGGGCUGGGCCGCCAAGGCAAGAUGGUGGACUACAGCGUGUGGG >hsa-mir-1182 MI0006275 (SEQ ID NO: 1406)GGGACUUGUCACUGCCUGUCUCCUCCCUCUCCAGCAGCGACUGGAUUCUGGAGUCCAUCUAGAGGGUCUUGGGAGGGAUGUGACUGUUGGGAAGCCC >hsa-mir-1183 MI0006276 (SEQ ID NO: 1407)AUUAUUCAAAUGCUCGGAGACACAGAACAUUAGAGAAGACAGGAGUUCACUGUAGGUGAUGGUGAGAGUGGGCAUGGAGCAGGAGUGCC >hsa-mir-1184-1 MI0006277 (SEQ ID NO: 1408)CUUGCAGAACGAGGUGAAGGAGGUGGUUCUGCUCAGCAGUCAACAGUGGCCACAUCUCCACCUGCAGCGACUUGAUGGCUUCCGUGUCCUUUUCGUGGG >hsa-mir-1184-2 MI0015971 (SEQ ID NO: 1409)CUUGCAGAACGAGGUGAAGGAGGUGGUUCUGCUCAGCAGUCAACAGUGGCCACAUCUCCACCUGCAGCGACUUGAUGGCUUCCGUGUCCUUUUCGUGGG >hsa-mir-1184-3 MI0015972 (SEQ ID NO: 1410)CUUGCAGAACGAGGUGAAGGAGGUGGUUCUGCUCAGCAGUCAACAGUGGCCACAUCUCCACCUGCAGCGACUUGAUGGCUUCCGUGUCCUUUUCGUGGG >hsa-mir-1185-l MI0003844 (SEQ ID NO: 1411)UUUGGUACUUGAAGAGAGGAUACCCUUUGUAUGUUCACUUGAUUAAUGGCGAAUAUACAGGGGGAGACUCUUAUUUGCGUAUCAAA >hsa-mir-1185-2 MI0003821 (SEQ ID NO: 1412)UUUGGUACUUAAAGAGAGGAUACCCUUUGUAUGUUCACUUGAUUAAUGGCGAAUAUACAGGGGGAGACUCUCAUUUGCGUAUCAAA >hsa-mir-1193 MI0014205 (SEQ ID NO: 1413)GUAGCUGAGGGGAUGGUAGACCGGUGACGUGCACUUCAUUUACGAUGUAGGUCACCCGUUUGACUAUCCACCAGCGC >hsa-mir-1197 MI0006656 (SEQ ID NO: 1414)ACUUCCUGGUAUUUGAAGAUGCGGUUGACCAUGGUGUGUACGCUUUAUUUGUGACGUAGGACACAUGGUCUACUUCUUCUCAAUAUCA >hsa-mir-1200 MI0006332 (SEQ ID NO: 1415)UGCUACUUCUCCUGAGCCAUUCUGAGCCUCAAUCACUUGCCAGAGAGAUUGGUUCAGGAAUUUGUCAGGGAUAGCC >hsa-mir-1202 MI0006334 (SEQ ID NO: 1416)CCUGCUGCAGAGGUGCCAGCUGCAGUGGGGGAGGCACUGCCAGGGCUGCCCACUCUGCUUAGCCAGCAGGUGCCAAGAACAGG >hsa-mir-1203 MI0006335 (SEQ ID NO: 1417)UCCUCCCCGGAGCCAGGAUGCAGCUCAAGCCACAGCAGGGUGUUUAGCGCUCUUCAGUGGCUCCAGAUUGUGGCGCUGGUGCAGG >hsa-mir-1204 MI0006337 (SEQ ID NO: 1418)ACCUCGUGGCCUGGUCUCCAUUAUUUGAGAUGAGUUACAUCUUGGAGGUGAGGACGUGCCUCGUGGU >hsa-mir-1205 MI0006338 (SEQ ID NO: 1419)GAAGGCCUCUGCAGGGUUUGCUUUGAGGUACUUCCUUCCUGUCAACCCUGUUCUGGAGUCUGU >hsa-mir-1206 MI0006339 (SEQ ID NO: 1420)CAGUGUUCAUGUAGAUGUUUAAGCUCUUGCAGUAGGUUUUUGCAAGCUAGUGAACGCUG >hsa-mir-1207 MI0006340 (SEQ ID NO: 1421)GCAGGGCUGGCAGGGAGGCUGGGAGGGGCUGGCUGGGUCUGGUAGUGGGCAUCAGCUGGCCCUCAUUUCUUAAGACAGCACUUCUGU >hsa-mir-1208 MI0006341 (SEQ ID NO: 1422)CACCGGCAGAAUCACUGUUCAGACAGGCGGAGACGGGUCUUUCUCGCCCUCUGAUGAGUCACCACUGUGGUGG >hsa-mir-1224 MI0003764 (SEQ ID NO: 1423)GUGAGGACUCGGGAGGUGGAGGGUGGUGCCGCCGGGGCCGGGCGCUGUUUCAGCUCGCUUCUCCCCCCACCUCCUCUCUCCUCAG >hsa-mir-1225 MI0006311 (SEQ ID NO: 1424)GUGGGUACGGCCCAGUGGGGGGGAGAGGGACACGCCCUGGGCUCUGCCCAGGGUGCAGCCGGACUGACUGAGCCCCUGUGCCGCCCCCAG >hsa-mir-1226 MI0006313 (SEQ ID NO: 1425)GUGAGGGCAUGCAGGCCUGGAUGGGGCAGCUGGGAUGGUCCAAAAGGGUGGCCUCACCAGCCCUGUGUUCCCUAG >hsa-mir-1227 MI0006316 (SEQ ID NO: 1426)GUGGGGCCAGGCGGUGGUGGGCACUGCUGGGGUGGGCACAGCAGCCAUGCAGAGCGGGCAUUUGACCCCGUGCCACCCUUUUCCCCAG >hsa-mir-1228 MI0006318 (SEQ ID NO: 1427)GUGGGCGGGGGCAGGUGUGUGGUGGGUGGUGGCCUGCGGUGAGCAGGGCCCUCACACCUGCCUCGCCCCCCAG >hsa-mir-1229 MI0006319 (SEQ ID NO: 1428)GUGGGUAGGGUUUGGGGGAGAGCGUGGGCUGGGGUUCAGGGACACCCUCUCACCACUGCCCUCCCACAG >hsa-mir-1231 MI0006321 (SEQ ID NO: 1429)GUCAGUGUCUGGGCGGACAGCUGCAGGAAAGGGAAGACCAAGGCUUGCUGUCUGUCCAGUCUGCCACCCUACCCUGUCUGUUCUUGCCACAG >hsa-mir-1233-1 MI0006323 (SEQ ID NO: 1430)GUGAGUGGGAGGCCAGGGCACGGCAGGGGGAGCUGCAGGGCUAUGGGAGGGGCCCCAGCGUCUGAGCCCUGUCCUCCCGCAG >hsa-mir-1233-2 MI0015973 (SEQ ID NO: 1431)GUGAGUGGGAGGCCAGGGCACGGCAGGGGGAGCUGCAGGGCUAUGGGAGGGGCCCCAGCGUCUGAGCCCUGUCCUCCCGCAG >hsa-mir-1234 MI0006324 (SEQ ID NO: 1432)GUGAGUGUGGGGUGGCUGGGGGGGGGGGGGGGGGGCCGGGGACGGCUUGGGCCUGCCUAGUCGGCCUGACCACCCACCCCACAG >hsa-mir-1236 MI0006326 (SEQ ID NO: 1433)GUGAGUGACAGGGGAAAUGGGGAUGGACUGGAAGUGGGCAGCAUGGAGCUGACCUUCAUCAUGGCUUGGCCAACAUAAUGCCUCUUCCCCUUGUCUCUCCAG >hsa-mir-1237 MI0006327 (SEQ ID NO: 1434)GUGGGAGGGCCCAGGCGCGGGCAGGGGUGGGGGUGGCAGAGCGCUGUCCCGGGGGCGGGGCCGAAGCGCGGCGACCGUAACUCCUUCUGCUCCGUCCCCCAG >hsa-mir-1238 MI0006328 (SEQ ID NO: 1435)GUGAGUGGGAGCCCCAGUGUGUGGUUGGGGCCAUGGCGGGUGGGCAGCCCAGCCUCUGAGCCUUCCUCGUCUGUCUGCCCCAG >hsa-mir-1243 MI0006373 (SEQ ID NO: 1436)CUAAAACUGGAUCAAUUAUAGGAGUGAAAUAAAGGUCCAUCUCCUGCCUAUUUAUUACUUUGCUUUGGUAAUAAAUCUAUUUUUAAAAGAACC >hsa-mir-1244-1 MI0006379 (SEQ ID NO: 1437)AUCUUAUUCCGAGCAUUCCAGUAACUUUUUUGUGUAUGUACUUAGCUGUACUAUAAGUAGUUGGUUUGUAUGAGAUGGUUAAAAA >hsa-mir-1244-2 MI0015974 (SEQ ID NO: 1438)AUCUUAUUCCGAGCAUUCCAGUAACUUUUUUGUGUAUGUACUUAGCUGUACUAUAAGUAGUUGGUUUGUAUGAGAUGGUUAAAAA >hsa-mir-1244-3 MI0015975 (SEQ ID NO: 1439)AUCUUAUUCCGAGCAUUCCAGUAACUUUUUUGUGUAUGUACUUAGCUGUACUAUAAGUAGUUGGUUUGUAUGAGAUGGUUAAAAA >hsa-mir-1245a MI0006380 (SEQ ID NO: 1440)AUUUAUGUAUAGGCCUUUAGAUCAUCUGAUGUUGAAUACUCUUUAAGUGAUCUAAAGGCCUACAUAUAAA >hsa-mir-1245b MI0017431 (SEQ ID NO: 1441)UUUAUAUGUAGGCCUUUAGAUCACUUAAAGAGUAUUCAACAUCAGAUGAUCUAAAGGCCUAUACAUAAA >hsa-mir-1246 MI0006381 (SEQ ID NO: 1442)UGUAUCCUUGAAUGGAUUUUUGGAGCAGGAGUGGACACCUGACCCAAAGGAAAUCAAUCCAUAGGCUAGCAAU >hsa-mir-1247 MI0006382 (SEQ ID NO: 1443)CCGCUUGCCUCGCCCAGCGCAGCCCCGGCCGCUGGGCGCACCCGUCCCGUUCGUCCCCGGACGUUGCUCUCUACCCCGGGAACGUCGAGACUGGAGCGCCCGAACUGAGCCACCUUCGCGGACCCCGAGAGCGGCG >hsa-mir-1248 MI0006383 (SEQ ID NO: 1444)UUUACCUUCUUGUAUAAGCACUGUGCUAAAAUUGCAGACACUAGGACCAUGUCUUGGUUUUUGCAAUAAUGCUAGCAGAGUACACACAAGAAGAAAAGUAACAGCA >hsa-mir-1249 MI0006384 (SEQ ID NO: 1445)GGGAGGAGGGAGGAGAUGGGCCAAGUUCCCUCUGGCUGGAACGCCCUUCCCCCCCUUCUUCACCUG >hsa-mir-1250 MI0006385 (SEQ ID NO: 1446)CUGUCCCGCUGGCCUGGCAGGUGACGGUGCUGGAUGUGGCCUUUUUGCCUUUUCUAAAGGCCACAUUUUCCAGCCCAUUCAACCUUCCAGAGCCCUCUGAAGUGGCCACAGGC >hsa-mir-1251 MI0006386 (SEQ ID NO: 1447)GUGGACUCUAGCUGCCAAAGGCGCUUCUCCUUCUGAACAGAGCGCUUUGCUCAGCCAGUGUAGACAUGGC >hsa-mir-1252 MI0006434 (SEQ ID NO: 1448)AGAAAGAAGGAAAUUGAAUUCAUUUAGAAAAGAGAAUUCCAAAUGAGCUUAAUUUCCUUUUUUCU >hsa-mir-1253 MI0006387 (SEQ ID NO: 1449)AGCAGCAAGAGAUAGAAUCCAAAAGAGAAGAAGAUCAGCCUGCAGAUGUGGACUGCUAAAUGCAGGCUGAUCUUCUCCCCUUUGGGAUUCUCUUAUGAGAAGCCA >hsa-mir-1254-1 MI0006388 (SEQ ID NO: 1450)GGUGGGAGGAUUGCUUGAGCCUGGAAGCUGGAGCCUGCAGUGAACUAUCAUUGUGCCACUGUACUCCAGCCUAGGCAACAAAAUGAAAUCCUGUCUA >hsa-mir-1254-2 MI0016747 (SEQ ID NO: 1451)CUGAGCCUGGAAGCUGGAGCCUGCAGUGAGCUAUGAUCAUGUCCCUGUACUCUAGCCUGGGCA >hsa-mir-1255a MI0006389 (SEQ ID NO: 14523)AUUGGAAAUCCUUUGAGUUGCUUCUCAAGGAUGAGCAAAGAAAGUAGAUUUUUUAGAUUCUAAAGAAACUAUCUUCUUUGCUCAUCCUUGAGAAGCAACUCCUUAUCCAUUAA >hsa-mir-1255b-1 MI0006435 (SEQ ID NO: 1453)UACGGAUGAGCAAAGAAAGUGGUUUCUUAAAAUGGAAUCUACUCUUUGUGAAGAUGCUGUGAA >hsa-mir-1255b-2 MI0006436 (SEQ ID NO: 1454)UCUUACGGAUGAGCAAAGAAAGUGGUUUGCGCCUCAAGAAACCACUUUCUUUGCUCAUCCAUAAGGA >hsa-mir-1256 MI0006390 (SEQ ID NO: 1455)AGUCAGCCUGUUGAAGCUUUGAAGCUUUGAUGCCAGGCAUUGACUUCUCACUAGCUGUGAAAGUCCUAGCUAAAGAGAAGUCAAUGCAUGACAUCUUGUUUCAAUAGAUGGCUGUUUCA >hsa-mir-1257 MI0006391 (SEQ ID NO: 1456)GCCCUGGGCUUGUGCUUGGGGAGUGAAUGAUGGGUUCUGACCCCCAUGCACCCCUGUGGGCCCCUGGCAUCACUGGCCCCAUCCUUCACCCCUGCCAACCACGCUUGCCCUGUGCCU >hsa-mir-1258 MI0006392 (SEQ ID NO: 1457)CUGUGGCUUCCACGACCUAAUCCUAACUCCUGCGAGUCCCUGGAGUUAGGAUUAGGUCGUGGAAGCCACAGGA >hsa-mir-1260a MI0006394 (SEQ ID NO: 1458)ACCUUUCCAGCUCAUCCCACCUCUGCCACCAAAACACUCAUCGCGGGGUCAGAGGGAGUGCCAAAAAAGGUAA >hsa-mir-1260b MI0014197 (SEQ ID NO: 1459)UCUCCGUUUAUCCCACCACUGCCACCAUUAUUGCUACUGUUCAGCAGGUGCUGCUGGUGGUGAUGGUGAUAGUCUGGUGGGGGCGGUGG >hsa-mir-1261 MI0006396 (SEQ ID NO: 1460)UGCUAUGGAUAAGGCUUUGGCUUAUGGGGAUAUUGUGGUUGAUCUGUUCUAUCCAGAUGACUGAAACUUUCUCCAUAGCAGC >hsa-mir-1262 MI0006397 (SEQ ID NO: 1461)AUCUACAAUGGUGAUGGGUGAAUUUGUAGAAGGAUGAAAGUCAAAGAAUCCUUCUGGGAACUAAUUUUUGGCCUUCAACAAGAAUUGUGAUAU >hsa-mir-1263 MI0006398 (SEQ ID NO: 1462)CUACCCCAAAAUAUGGUACCCUGGCAUACUGAGUAUUUUAAUACUGGCAUACUCAGUAUGCCAUGUUGCCAUAUUUUGGGGUAGCA >hsa-mir-1264 MI0003758 (SEQ ID NO: 1463)AGGUCCUCAAUAAGUAUUUGUUGAAAGAAUAAAUAAACCAACAAGUCUUAUUUGAGCACCUGUUAUGUG >hsa-mir-1265 MI0006401 (SEQ ID NO: 1464)AUGGUUUGGGACUCAGGAUGUGGUCAAGUGUUGUUAAGGCAUGUUCAGGAACAAUACUUGACCACAUUUUGAAUUCCAAACCAUAU >hsa-mir-1266 MI0006403 (SEQ ID NO: 1465)ACAGGUAGUGUCCCUCAGGGCUGUAGAACAGGGCUGGGAUUACUAAAGCCCUGUUCUAUGCCCUGAGGGACACUGAGCAUGUCA >hsa-mir-1267 MI0006404 (SEQ ID NO: 1466)CUCCCAAAUCUCCUGUUGAAGUGUAAUCCCCACCUCCAGCAUUGGGGAUUACAUUUCAACAUGAGAUUUGGAUGAGGA >hsa-mir-1268a MI0006405 (SEQ ID NO: 1467)UAGCCGGGCGUGGUGGUGGGGGCCUGUGGUCCCAGCUACUUUGGAGGCUGAG >hsa-mir-1268b MI0016748 (SEQ ID NO: 1468)ACCCGGGCGUGGUGGUGGGGGUGGGUGCCUGUAAUUCCAGCUAGUUGGGA >hsa-mir-1269a MI0006406 (SEQ ID NO: 1469)UGGAUUGCCUAGACCAGGGAAGCCAGUUGGCAUGGCUCAGUCCAAGUCUGACCACCUGAGGAAUGCCUGGACUGAGCCGUGCUACUGGCUUCCCUGGUCUCCAGC >hsa-mir-1269b MI0016888 (SEQ ID NO: 1470)UGAGGUUUCUGGACUGAGCCAUGCUACUGGCUUCUCUGGUUCUCCAGCUUACAGAUGGCUUAUCAUGGGACCUCU >hsa-mir-1270-1 MI0006407 (SEQ ID NO: 1471)CACAGAGUUAUACUGGAGAUAUGGAAGAGCUGUGUUGGGUAUAAGUAACAGGCUUUUCUUUAUCUUCUAUGUGGCUCUUUGCA >hsa-mir-1270-2 MI0015976 (SEQ ID NO: 1472)CACAGAGUUAUACUGGAGAUAUGGAAGAGCUGUGUUGGGUAUAAGUAACAGGCUUUUCUUUAUCUUCUAUGUGGCUCUUUGCA >hsa-mir-1271 MI0003814 (SEQ ID NO: 1473)CACCCAGAUCAGUGCUUGGCACCUAGCAAGCACUCAGUAAAUAUUUGUUGAGUGCCUGCUAUGUGCCAGGCAUUGUGCUGAGGGCU >hsa-mir-1272 MI0006408 (SEQ ID NO: 1474)CCAGAUCAGAUCUGGGUGCGAUGAUGAUGGCAGCAAAUUCUGAAAACGUGCUCAGUGUCUUUAUAACAGGAAAGCCGUAAACUUAGAAAUGUAGGCUGCAGCUCGUGUGCUCUGUGGUCUGGGCUGGUA >hsa-mir-1273a MI0006409 (SEQ ID NO: 1475)UGAGGCAGGAGAAUUGCUUGAACCCGGGUGGUGGAGGUUGCAGUGAGCCAAGAUUGCGCCACUGCACUCCAGCCUGGGCGACAAAGCAAGACUCUUUCUUGGA >hsa-mir-1273c MI0014171 (SEQ ID NO: 1476)UGCAGCCUGGGCGACAAAACGAGACCCUGUCUUUUUUUUUUUCUGAGACAGAGUCUCGUUCUGUUGCCCAAGCUGGA >hsa-mir-1273d MI0014254 (SEQ ID NO: 1477)GAAUCGCUUGAACCCAUGAGGUUGAGGCUGCAGUGAGCCAAGAUCGUGCCACUGCACUUCAGCCUGGGUGACAAGAGCGAAACUUC >hsa-mir-1273e MI0016059 (SEQ ID NO: 1478)UGAGGCAGGAGAAUUGCUUGAACCCAGGAAGUGGAGGCUGCAGUGAGCCGAGAUCGAGCCACUGUACUCCAGCCUGGGUGACACAGCGAGACUCCAGUCUCA >hsa-mir-1273f MI0018002 (SEQ ID NO: 1479)AGGUGGGAGGAUUGCUUGAGCCUGGGAGAUGGAGGUUGCAGUGAGCUGAGAUCACGCAACUGCACCCCCAGCCUGGGCCAUAGAGUCAGUCCUUGUCUC >hsa-mir-1273g MI0018003 (SEQ ID NO: 1480)GAGGUGGGAGGAUUGCUUGAGUCAGGGUGGUUGAGGCUGCAGUAAGUUGUGAUCAUACCACUGCACUCCAGCCUGAGUGACAGAGCAAGACCUUGUCUCA >hsa-mir-1275 MI0006415 (SEQ ID NO: 1481)CCUCUGUGAGAAAGGGUGUGGGGGAGAGGCUGUCUUGUGUCUGUAAGUAUGCCAAACUUAUUUUCCCCAAGGCAGAGGGA >hsa-mir-1276 MI0006416 (SEQ ID NO: 1482)CCCCAGCUAGGUAAAGAGCCCUGUGGAGACACCUGGAUUCAGAGAACAUGUCUCCACUGAGCACUUGGGCCUUGAUGGCGGCU >hsa-mir-1277 MI0006419 (SEQ ID NO: 1483)ACCUCCCAAAUAUAUAUAUAUAUGUACGUAUGUGUAUAUAAAUGUAUACGUAGAUAUAUAUGUAUUUUUGGUGGGUUU >hsa-mir-1278 MI0006425 (SEQ ID NO: 1484)AUUUGCUCAUAGAUGAUAUGCAUAGUACUCCCAGAACUCAUUAAGUUGGUAGUACUGUGCAUAUCAUCUAUGAGCGAAUAG >hsa-mir-1279 MI0006426 (SEQ ID NO: 1485)AUAUUCACAAAAAUUCAUAUUGCUUCUUUCUAAUGCCAAGAAAGAAGAGUAUAAGAACUUCC >hsa-mir-1280 MI0006437 (SEQ ID NO: 1486)UCUGUCCCACCGCUGCCACCCUCCCCUCUGCCUCAGUGUGCCAGGCAUCAGCACUCACUCACAGAGGCAGGCUGGAUGGCGGGUGGGACAACAG >hsa-mir-1281 MI0006428 (SEQ ID NO: 1487)AGGGGGCACCGGGAGGAGGUGAGUGUCUCUUGUCGCCUCCUCCUCUCCCCCCUU >hsa-mir-1282 MI0006429 (SEQ ID NO: 1488)CCUUCUUCUCGUUUGCCUUUUUCUGCUUCUGCUGCAUGAUCUCCGAGUCCCUGGGGGUAGAGAUGAUGGGGCACUGGGAGGUACCAGAGGGCAAAAAGGAC >hsa-mir-1283-1 MI0003832 (SEQ ID NO: 1489)CUCAAGCUAUGAGUCUACAAAGGAAAGCGCUUUCUGUUGUCAGAAAGAAGAGAAAGCGCUUCCCUUUUGAGGGUUACGGUUUGAGAA >hsa-mir-1283-2 MI0006430 (SEQ ID NO: 1490)CUCAAGCUGUGAGUCUACAAAGGAAAGCGCUUUCUGUUGUCUGAAAGAAAAGAAAUCGCUUCCCUUUGGAGUGUUACGGUUUGAGAA >hsa-mir-1284 MI0006431 (SEQ ID NO: 1491)AUUUUGAUAUAUAAGCCAGUUUAAUGUUUUCUAUACAGACCCUGGCUUUUCUUAAAUUUUAUAUAUUGGAAAGCCCAUGUUUGUAUUGGAAACUGCUGGUUUCUUUCAUACUGAAAAUCU >hsa-mir-1285-1 MI0006346 (SEQ ID NO: 1492)UGUAGAGAUAGGAUCUCACUUUGUUGCCCAGGCUGGUCUCAAACUCCUGGUCUGGGCAACAAAGUGAGACCUUAUCUCUACAAG >hsa-mir-1285-2 MI0006347 (SEQ ID NO: 1493)UUUGGGAGGCCGAGGCUGGUGCAUCACUUGAGCCCAGCAAUUUGAGACCAAUCUGGGCAACAAAGUGAGACCUCCGUCUCUACAAAGA >hsa-mir-1286 MI0006348 (SEQ ID NO: 1494)UGUCCUCUGGGGACUCAGCUUGCUCUGGCUGCUGGAUUGAAUUAGCUGCAGGACCAAGAUGAGCCCUUGGUGGAGACA >hsa-mir-1287 MI0006349 (SEQ ID NO: 1495)GUUGUGCUGUCCAGGUGCUGGAUCAGUGGUUCGAGUCUGAGCCUUUAAAAGCCACUCUAGCCACAGAUGCAGUGAUUGGAGCCAUGACAA >hsa-mir-1288 MI0006432 (SEQ ID NO: 1496)GAGGGUGUUGAUCAGCAGAUCAGGACUGUAACUCACCAUAGUGGUGGACUGCCCUGAUCUGGAGACCACUGCCUU >hsa-mir-1289-1 MI0006350 (SEQ ID NO: 1497)UUCUCAAUUUUUAGUAGGAAUUAAAAACAAAACUGGUAAAUGCAGACUCUUGGUUUCCACCCCCAGAGAAUCCCUAAACCGGGGGUGGAGUCCAGGAAUCUGCAUUUUAGAAAGUACCCAGGGUGAUUCUGAUAAUUGGGAACA >hsa-mir-1289-2 MI0006351 (SEQ ID NO: 1498)CCACGGUCCUAGUUAAAAAGGCACAUUCCUAGACCCUGCCUCAGAACUACUGAACAGAGUCACUGGGUGUGGAGUCCAGGAAUCUGCAUUUUUACCCCUAUCGCCCCCGCC >hsa-mir-1290 MI0006352 (SEQ ID NO: 1499)GAGCGUCACGUUGACACUCAAAAAGUUUCAGAUUUUGGAACAUUUCGGAUUUUGGAUUUUUGGAUCAGGGAUGCUCAA >hsa-mir-1291 MI0006353 (SEQ ID NO: 1500)GGUAGAAUUCCAGUGGCCCUGACUGAAGACCAGCAGUUGUACUGUGGCUGUUGGUUUCAAGCAGAGGCCUAAAGGACUGUCUUCCUG >hsa-mir-1292 MI0006433 (SEQ ID NO: 1501)CCUGGGAACGGGUUCCGGCAGACGCUGAGGUUGCGUUGACGCUCGCGCCCCGGCUCCCGUUCCAGG >hsa-mir-1293 MI0006355 (SEQ ID NO: 1502)AGGUUGUUCUGGGUGGUCUGGAGAUUUGUGCAGCUUGUACCUGCACAAAUCUCCGGACCACUUAGUCUUUA >hsa-mir-1294 MI0006356 (SEQ ID NO: 1503)CACCUAAUGUGUGCCAAGAUCUGUUCAUUUAUGAUCUCACCGAGUCCUGUGAGGUUGGCAUUGUUGUCUGGCAUUGUCUGAUAUACAACAGUGCCAACCUCACAGGACUCAGUGAGGUGAAACUGAGGAUUAGGAAGGUGUA >hsa-mir-1295a MI0006357 (SEQ ID NO: 1504)AGGACAUUUUGCCCAGAUCCGUGGCCUAUUCAGAAAUGUGGCCUGUGAUUAGGCCGCAGAUCUGGGUGAAAUGUCCUCC >hsa-mir-1295b MI0019146 (SEQ ID NO: 1505)CACCCAGAUCUGCGGCCUAAUCACAGGCCACAUUUCUGAAUAGGCCACGGAUCUGGGCAA >hsa-mir-1296 MI0003780 (SEQ ID NO: 1506)ACCUACCUAACUGGGUUAGGGCCCUGGCUCCAUCUCCUUUAGGAAAACCUUCUGUGGGGAGUGGGGCUUCGACCCUAACCCAGGUGGGCUGU >hsa-mir-1297 MI0006358 (SEQ ID NO: 1507)UGUUUAUCUCUAGGGUUGAUCUAUUAGAAUUACUUAUCUGAGCCAAAGUAAUUCAAGUAAUUCAGGUGUAGUGAAAC >hsa-mir-1298 MI0003938 (SEQ ID NO: 1508)AGACGAGGAGUUAAGAGUUCAUUCGGCUGUCCAGAUGUAUCCAAGUACCCUGUGUUAUUUGGCAAUAAAUACAUCUGGGCAACUGACUGAACUUUUCACUUUUCAUGACUCA >hsa-mir-1299 MI0006359 (SEQ ID NO: 1509)CCUCAUGGCAGUGUUCUGGAAUCCUACGUGAGGGACAAUCAUUCAGACCCACGUAGCAGUGUUCUGGAAUUCUGUGUGAGGGA >hsa-mir-1301 MI0003815 (SEQ ID NO: 1510)GGAUUGUGGGGGGUCGCUCUAGGCACCGCAGCACUGUGCUGGGGAUGUUGCAGCUGCCUGGGAGUGACUUCACACAGUCCUC >hsa-mir-1302-1 MI0006362 (SEQ ID NO: 1511)CAGAAAGCCCAGUUAAAUUUGAAUUUCAAGUAAACAAUGAAUAAUUGUGUAUGUAAGAAUAUCCCAUACAAUAUUUGGGACAUACUUAUGCUAAAAAUUAUUCCUUGCUUAUCUGAAAUUCAAAUGUAACUAGGAUUCCUGUA >hsa-mir-1302-10 MI0015979 (SEQ ID NO: 1512)GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUGUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAGAAUUAAGCAUUUUA >hsa-mir-1302-11 MI0015980 (SEQ ID NO: 1513)GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUGUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAGAAUUAAGCAUUUUA >hsa-mir-1302-2 MI0006363 (SEQ ID NO: 1514)GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUGUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAGAAUUAAGCAUUUUA >hsa-mir-1302-3 MI0006364 (SEQ ID NO: 1515)GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUUUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAAAAUUAAGCAUUUUA >hsa-mir-1302-4 MI0006365 (SEQ ID NO: 1516)AAUGCAGAAGCACAGCUAAAAUUUGAAUUUCAGAUAAACAAAUUUUUCUUAGAAUAAGUAUGUCUCCAUGCAACAUUUGGGACAUACUUAUGCUAAAAUAUUAUUUGUGUUUCAUCUGAAAUUCAAAUUCAACUGGACAUCCUGUAUUUU >hsa-mir-1302-5 MI0006366 (SEQ ID NO: 1517)UGCCCGGCCUCCCAUUAAAUUGGUUUUUCAGACAAAUCACAAAUUUGUUUAGGUAUAAGUAUAUCCCAUGUAAUCUUUGGGACAUACUUAUGCUAAAAUAAUUGUUCCUUGUUGAUUGGAAAUUUUAAUUUUAAUUAGGUGUCCUGUAUU >hsa-mir-1302-6 MI0006367 (SEQ ID NO: 1518)AACAAAUAAUUUGGUAAUAUAUGUAUGGCCCACACAAUAUUUAGGACAACAAUAUUUGGGACAUACUUAUGCUAAAAAAGUAUUUGUUGA >hsa-mir-1302-7 MI0006368 (SEQ ID NO: 1519)ACAACAUGUUUUUAGGACAUGUAUGUCUGGUGCAAUAAUUGGGACAUACUUAUGCUAAAAAAAUUAGUGUUC >hsa-mir-1302-8 MI0006369 (SEQ ID NO: 1520)CCCAUUUAAACUUGAAUUUCAUAUAAACACCGUAAUUUUCAGCAUUAGUGUAUCACAUGCAGUAUUUGGGACAUACUUAUGCUAAAAAAUUAGGUGGUGUUGAUCUGAAAUUCCAGUGUAGAUGGGCA >hsa-mir-1302-9 MI0015978 (SEQ ID NO: 1521)GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUGUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAGAAUUAAGCAUUUUA >hsa-mir-1303 MI0006370 (SEQ ID NO: 1522)GGCUGGGCAACAUAGCGAGACCUCAACUCUACAAUUUUUUUUUUUUUAAAUUUUAGAGACGGGGUCUUGCUCUGUUGCCAGGCUUU >hsa-mir-1304 MI0006371 (SEQ ID NO: 1523)AAACACUUGAGCCCAGCGGUUUGAGGCUACAGUGAGAUGUGAUCCUGCCACAUCUCACUGUAGCCUCGAACCCCUGGGCUCAAGUGAUUCA >hsa-mir-1305 MI0006372 (SEQ ID NO: 1524)AAGAUCCUGCUGUUUCUACCAUUAGUUUUGAAUGUUUAUUGUAAAGAUACUUUUCAACUCUAAUGGGAGAGACAGCAGGAUUCUCC >hsa-mir-1306 MI0006443 (SEQ ID NO: 1525)GUGAGCAGUCUCCACCACCUCCCCUGCAAACGUCCAGUGGUGCAGAGGUAAUGGACGUUGGCUCUGGUGGUGAUGGACAGUCCGA >hsa-mir-1307 MI0006444 (SEQ ID NO: 1526)CAUCAAGACCCAGCUGAGUCACUGUCACUGCCUACCAAUCUCGACCGGACCUCGACCGGCUCGUCUGUGUUGCCAAUCGACUCGGCGUGGCGUCGGUCGUGGUAGAUAGGCGGUCAUGCAUACGAAUUUUCAGCUCUUGUUCUGGUGAC >hsa-mir-1321 MI0006652 (SEQ ID NO: 1527)ACAUUAUGAAGCAAGUAUUAUUAUCCCUGUUUUACAAAUAAGGAAAUAAACUCAGGGAGGUGAAUGUGAUCAAAGAUAG >hsa-mir-1322 MI0006653 (SEQ ID NO: 1528)AGUAUCAUGAAUUAGAAACCUACUUAUUACAUAGUUUACAUAAGAAGCGUGAUGAUGCUGCUGAUGCUGUA >hsa-mir-1323 MI0003786 (SEQ ID NO: 1529)ACUGAGGUCCUCAAAACUGAGGGGCAUUUUCUGUGGUUUGAAAGGAAAGUGCACCCAGUUUUGGGGAUGUCAA >hsa-mir-1324 MI0006657 (SEQ ID NO: 1530)CCUGAAGAGGUGCAUGAAGCCUGGUCCUGCCCUCACUGGGAACCCCCUUCCCUCUGGGUACCAGACAGAAUUCUAUGCACUUUCCUGGAGGCUCCA >hsa-mir-1343 MI0017320 (SEQ ID NO: 1531)GCUGGCGUCGGUGCUGGGGAGCGGCCCCCGGGUGGGCCUCUGCUCUGGCCCCUCCUGGGGCCCGCACUCUCGCUCUGGGCCCGC >hsa-mir-1468 MI0003782 (SEQ ID NO: 1532)GGUGGGUGGUUUCUCCGUUUGCCUGUUUCGCUGAUGUGCAUUCAACUCAUUCUCAGCAAAAUAAGCAAAUGGAAAAUUCGUCCAUC >hsa-mir-1469 MI0007074 (SEQ ID NO: 1533)CUCGGCGCGGGGCGCGGGCUCCGGGUUGGGGCGAGCCAACGCCGGGG >hsa-mir-1470 MI0007075 (SEQ ID NO: 1534)GCCCUCCGCCCGUGCACCCCGGGGCAGGAGACCCCGCGGGACGCGCCGAGGUAGGGGGGAC >hsa-mir-1471 MI0007076 (SEQ ID NO: 1535)GCCCGCGUGUGGAGCCAGGUGUAGAGGCGGAGCACAGCUGGCUCUAAUUUGAGGGGC >hsa-mir-1537 MI0007258 (SEQ ID NO: 1536)ACAGCUGUAAUUAGUCAGUUUUCUGUCCUGUCCACACAGAAAACCGUCUAGUUACAGUUGU >hsa-mir-1538 MI0007259 (SEQ ID NO: 1537)GGGAACAGCAGCAACAUGGGCCUCGCUUCCUGCCGGCGCGGCCCGGGCUGCUGCUGUUCCU >hsa-mir-1539 MI0007260 (SEQ ID NO: 1538)GGCUCUGCGGCCUGCAGGUAGCGCGAAAGUCCUGCGCGUCCCAGAUGCCC >hsa-mir-1587 MI0016905 (SEQ ID NO: 1539)UUUGGGCUGGGCUGGGUUGGGCAGUUCUUCUGCUGGACUCACCUGUGACCAGC >hsa-mir-1825 MI0008193 (SEQ ID NO: 1540)AGAGACUGGGGUGCUGGGCUCCCCUAGACUAGGACUCCAGUGCCCUCCUCUCC >hsa-mir-1827 MI0008195 (SEQ ID NO: 1541)UCAGCAGCACAGCCUUCAGCCUAAAGCAAUGAGAAGCCUCUGAAAGGCUGAGGCAGUAGAUUGAAU >hsa-mir-1908 MI0008329 (SEQ ID NO: 1542)CGGGAAUGCCGCGGCGGGGACGGCGAUUGGUCCGUAUGUGUGGUGCCACCGGCCGCCGGCUCCGCCCCGGCCCCCGCCCC >hsa-mir-1909 MI0008330 (SEQ ID NO: 1543)CAUCCAGGACAAUGGUGAGUGCCGGUGCCUGCCCUGGGGCCGUCCCUGCGCAGGGGCCGGGUGCUCACCGCAUCUGCCCC >hsa-mir-1910 MI0008331 (SEQ ID NO: 1544)UGUCCCUUCAGCCAGUCCUGUGCCUGCCGCCUUUGUGCUGUCCUUGGAGGGAGGCAGAAGCAGGAUGACAAUGAGGGCAA >hsa-mir-1911 MI0008332 (SEQ ID NO: 1545)UCGGCAUCUGCUGAGUACCGCCAUGUCUGUUGGGCAUCCACAGUCUCCCACCAGGCAUUGUGGUCUCCGCUGACGCUUUG >hsa-mir-1912 MI0008333 (SEQ ID NO: 1546)CUCUAGGAUGUGCUCAUUGCAUGGGCUGUGUAUAGUAUUAUUCAAUACCCAGAGCAUGCAGUGUGAACAUAAUAGAGAUU >hsa-mir-1913 MI0008334 (SEQ ID NO: 1547)ACCUCUACCUCCCGGCAGAGGAGGCUGCAGAGGCUGGCUUUCCAAAACUCUGCCCCCUCCGCUGCUGCCAAGUGGCUGGU >hsa-mir-1914 MI0008335 (SEQ ID NO: 1548)CGUGUGAGCCCGCCCUGUGCCCGGCCCACUUCUGCUUCCUCUUAGCGCAGGAGGGGUCCCGCACUGGGAGGGGCCCUCAC >hsa-mir-1915 MI0008336 (SEQ ID NO: 1549)UGAGAGGCCGCACCUUGCCUUGCUGCCCGGGCCGUGCACCCGUGGGCCCCAGGGCGACGCGGCGGGGGCGGCCCUAGCGA >hsa-mir-1972-1 MI0009982 (SEQ ID NO: 1550)UAUAGGCAUGUGCCACCACACCUGGCUUAAAUGUGUCAUUUAAAAAUUCAGGCCAGGCACAGUGGCUCAUGCCUGUA >hsa-mir-1972-2 MI0015977 (SEQ ID NO: 1551)UAUAGGCAUGUGCCACCACACCUGGCUUAAAUGUGUCAUUUAAAAAUUCAGGCCAGGCACAGUGGCUCAUGCCUGUA >hsa-mir-1973 MI0009983 (SEQ ID NO: 1552)UAUGUUCAACGGCCAUGGUAUCCUGACCGUGCAAAGGUAGCAUA >hsa-mir-1976 MI0009986 (SEQ ID NO: 1553)GCAGCAAGGAAGGCAGGGGUCCUAAGGUGUGUCCUCCUGCCCUCCUUGCUGU >hsa-mir-2052 MI0010486 (SEQ ID NO: 1554)CUGUUUUGAUAACAGUAAUGUCCCUUUAGUUCAAAGUUACCAGCUAUCAAAACAA >hsa-mir-2053 MI0010487 (SEQ ID NO: 1555)CUUGCCAUGUAAAUACAGAUUUAAUUAACAUUUGCAACCUGUGAAGAUGCAAAACUUUAAGUGUUAAUUAAACCUCUAUUUACAUAGCAAG >hsa-mir-2054 MI0010488 (SEQ ID NO: 1556)CUGUAAUAUAAAUUUAAUUUAUUCUCUAUCAUUAAAAAAUGUAUUACAG >hsa-mir-2110 MI0010629 (SEQ ID NO: 1557)CAGGGGUUUGGGGAAACGGCCGCUGAGUGAGGCGUCGGCUGUGUUUCUCACCGCGGUCUUUUCCUCCCACUCUUG >hsa-mir-2113 MI0003939 (SEQ ID NO: 1558)UUUUCAAAGCAAUGUGUGACAGGUACAGGGACAAAUCCCGUUAAUAAGUAAGAGGAUUUGUGCUUGGCUCUGUCACAUGCCACUUUGAAAA >hsa-mir-2114 MI0010633 (SEQ ID NO: 1559)CCUCCAUGCUCCUAGUCCCUUCCUUGAAGCGGUCGGAUAAUCACAUGACGAGCCUCAAGCAAGGGACUUCAAGCUGGUGG >hsa-mir-2115 MI0010634 (SEQ ID NO: 1560)ACUGUCAUCCCACUGCUUCCAGCUUCCAUGACUCCUGAUGGAGGAAUCACAUGAAUUCAUCAGAAUUCAUGGAGGCUAGAAGCAGUAUGAGGAUCAUUUA >hsa-mir-2116 MI0010635 (SEQ ID NO: 1561)GACCUAGGCUAGGGGUUCUUAGCAUAGGAGGUCUUCCCAUGCUAAGAAGUCCUCCCAUGCCAAGAACUCCCAGACUAGGA >hsa-mir-2117 MI0010636 (SEQ ID NO: 1562)GCUCUGAUUUACUUCUGUCCGGCAUGGUGAACAGCAGGAUUGGCUGUAGCUGUUCUCUUUGCCAAGGACAGAUCUGAUCU >hsa-mir-2276 MI0011282 (SEQ ID NO: 1563)GUGUUCUUCCAGUCCGCCCUCUGUCACCUUGCAGACGGCUUUCUCUCCGAAUGUCUGCAAGUGUCAGAGGCGAGGAGUGGCAGCUGCAU >hsa-mir-2277 MI0011284 (SEQ ID NO: 1564)GUGCUUCCUGCGGGCUGAGCGCGGGCUGAGCGCUGCCAGUCAGCGCUCACAUUAAGGCUGACAGCGCCCUGCCUGGCUCGGCCGGCGAAGCUC >hsa-mir-2278 MI0011285 (SEQ ID NO: 1565)GUGCUGCAGGUGUUGGAGAGCAGUGUGUGUUGCCUGGGGACUGUGUGGACUGGUAUCACCCAGACAGCUUGCACUGACUCCAGACCCUGCCGUCAU >hsa-mir-2355 MI0015873 (SEQ ID NO: 1566)CAGACGUGUCAUCCCCAGAUACAAUGGACAAUAUGCUAUUAUAAUCGUAUGGCAUUGUCCUUGCUGUUUGGAGAUAAUACUGCUGAC >hsa-mir-2392 MI0016870 (SEQ ID NO: 1567)AUGGUCCCUCCCAAUCCAGCCAUUCCUCAGACCAGGUGGCUCCCGAGCCACCCCAGGCUGUAGGAUGGGGGUGAGAGGUGCUAG >hsa-mir-2467 MI0017432 (SEQ ID NO: 1568)GGACAGGCACCUGAGGCUCUGUUAGCCUUGGCUCUGGGUCCUGCUCCUUAGAGCAGAGGCAGAGAGGCUCAGGGUCUGUCU >hsa-mir-2681 MI0012062 (SEQ ID NO: 1569)GCCCCCUUUUCACGCAUUUGUGUUUUACCACCUCCAGGAGACUGCCCAAAGACUCUUCAGUAUCAUGGAGUUGGUAAAGCACAGAUGCAUGAAUAAUUCAACGUG >hsa-mir-2682 MI0012063 (SEQ ID NO: 1570)ACCUUCCUGAAAGAGGUUGGGGCAGGCAGUGACUGUUCAGACGUCCAAUCUCUUUGGGACGCCUCUUCAGCGCUGUCUUCCCUGCCUCUGCCUUUAGGACGAGUCUCAAA >hsa-mir-2861 MI0013006 (SEQ ID NO: 1571)GGCGCCUCUGCAGCUCCGGCUCCCCCUGGCCUCUCGGGAACUACAAGUCCCAGGGGGCCUGGCGGUGGGCGGCGGGCGGAAGAGGCGGGG >hsa-mir-2909 MI0013083 (SEQ ID NO: 1572)GGUGUUAGGGCCAACAUCUCUUGGUCUUUCCCCUGUGGUCCCAAGAUGGCUGUUGCAACUUAACGCCAU >hsa-mir-2964a MI0017299 (SEQ ID NO: 1573)GGAGCUCAGCCACAGAUGUCCAGCCACAAUUCUCGGUUGGCCGCAGACUCGUACAAGAAUUGCGUUUGGACAAUCAGUGGCGAAGCCC >hsa-mir-3064 MI0017375 (SEQ ID NO: 1574)GGUCUGGCUGUUGUGGUGUGCAAAACUCCGUACAUUGCUAUUUUGCCACACUGCAACACCUUACAG >hsa-mir-3065 MI0014228 (SEQ ID NO: 1575)CUGCCCUCUUCAACAAAAUCACUGAUGCUGGAGUCGCCUGAGUCAUCACUCAGCACCAGGAUAUUGUUGGAGAGGACAG >hsa-mir-3074 MI0014181 (SEQ ID NO: 1576)GCUCGACUCCUGUUCCUGCUGAACUGAGCCAGUGUGUAAAAUGAGAACUGAUAUCAGCUCAGUAGGCACCGGAGGGCGGGU >hsa-mir-3115 MI0014127 (SEQ ID NO: 1577)UCUGAAUAUGGGUUUACUAGUUGGUGGUGAAUUCAUGAGUCGCCAACUAUUAGGCCUUUAUGUCCAGA >hsa-mir-3116-1 MI0014128 (SEQ ID NO: 1578)CUUUAUUGAGUCCCUACUAUGUUCCAGGCACUGGGUAUCGUAGGUGCCUGGAACAUAGUAGGGACUCAAUAAAG >hsa-mir-3116-2 MI0014129 (SEQ ID NO: 1579)UAUUGAGUCCCUACUAUGUUCCAGGCACCUACGAUACCCAGUGCCUGGAACAUAGUAGGGACUCAAUA >hsa-mir-3117 MI0014130 (SEQ ID NO: 1580)CCCUAAAGGGCCAGACACUAUACGAGUCAUAUAAGGGAAGGCAUUAUAGGACUCAUAUAGUGCCAGGUGUUUUGUGGG >hsa-mir-3118-1 MI0014131 (SEQ ID NO: 1581)CACACUACAAUAAUUUUCAUAAUGCAAUCACACAUAAUCACUAUGUGACUGCAUUAUGAAAAUUCUUGUAGUGUG >hsa-mir-3118-2 MI0014132 (SEQ ID NO: 1582)ACACUACAAUAAUUUUCAUAAUGCAAUCACACAUAAUCACUAUGUGACUGCAUUAUGAAAAUUCUUGUAGUGU >hsa-mir-3118-3 MI0014133 (SEQ ID NO: 1583)CACACUACAAUAAUUUUCAUAAUGCAAUCACACAUAAUCACUAUGUGACUGCAUUAUGAAAAUUCUUGUAGUGUG >hsa-mir-3118-4 MI0014207 (SEQ ID NO: 1584)CAUACUACAAUAAUUUUCAUAAUGCAAUCACACACAAUCACCGUGUGACUGCAUUAUGAAAAUUCUUCUAGUGUG >hsa-mir-3118-5 MI0014243 (SEQ ID NO: 1585)CACACAUACAAUAAUAUUCAUAAUGCAAUCACACACAAUCACCAUGUGACUGCAUUAUGAAAAUUCUUCUAGUGUG >hsa-mir-3118-6 MI0015981 (SEQ ID NO: 1586)CAUACUACAAUAAUUUUCAUAAUGCAAUCACACACAAUCACCGUGUGACUGCAUUAUGAAAAUUCUUCUAGUGUG >hsa-mir-3119-1 MI0014134 (SEQ ID NO: 1587)AUUAACUCUGGCUUUUAACUUUGAUGGCAAAGGGGUAGCUAAACAAUCUAUGUCUUUGCCAUCAAAGUUAAAAGCCAUAGUUAAU >hsa-mir-3119-2 MI0014135 (SEQ ID NO: 1588)AUUAACUAUGGCUUUUAACUUUGAUGGCAAAGACAUAGAUUGUUUAGCUACCCCUUUGCCAUCAAAGUUAAAAGCCAGAGUUAAU >hsa-mir-3120 MI0014136 (SEQ ID NO: 1589)GUCAUGUGACUGCCUGUCUGUGCCUGCUGUACAGGUGAGCGGAUGUUCUGCACAGCAAGUGUAGACAGGCAGACACAUGAC >hsa-mir-3121 MI0014137 (SEQ ID NO: 1590)AAAUGGUUAUGUCCUUUGCCUAUUCUAUUUAAGACACCCUGUACCUUAAAUAGAGUAGGCAAAGGACAGAAACAUUU >hsa-mir-3122 MI0014138 (SEQ ID NO: 1591)ACCAGCUCUGUUGGGACAAGAGGACGGUCUUCUUUUGGAAGGAAGACCAUCAUCUUGUCCGAAGAGAGCUGGU >hsa-mir-3123 MI0014139 (SEQ ID NO: 1592)AUGGAUUUGAUUGAAUGAUUCUCCCAUUUCCACAUGGAGAGUGGAGCCCAGAGAAUUGUUUAAUCAUGUAUCCAU >hsa-mir-3124 MI0014140 (SEQ ID NO: 1593)GCGGGCUUCGCGGGCGAAGGCAAAGUCGAUUUCCAAAAGUGACUUUCCUCACUCCCGUGAAGUCGGC >hsa-mir-3125 MI0014142 (SEQ ID NO: 1594)GAGAAUGGGUAGAGGAAGCUGUGGAGAGAACUCACGGUGCCUGUGGUUCGAGAUCCCCGCCUUCCUCCUCCUUUCCUC >hsa-mir-3126 MI0014143 (SEQ ID NO: 1595)AUGAUUAUAUGAGGGACAGAUGCCAGAAGCACUGGUUAUGAUUUGCAUCUGGCAUCCGUCACACAGAUAAUUAU >hsa-mir-3127 MI0014144 (SEQ ID NO: 1596)GGCCAGGCCCAUCAGGGCUUGUGGAAUGGGAAGGAGAAGGGACGCUUCCCCUUCUGCAGGCCUGCUGGGUGUGGCU >hsa-mir-3128 MI0014145 (SEQ ID NO: 1597)UUCCUCUGGCAAGUAAAAAACUCUCAUUUUCCUUAAAAAAUGAGAGUUUUUUACUUGCAAUAGGAA >hsa-mir-3129 MI0014146 (SEQ ID NO: 1598)GUACUUGGGCAGUAGUGUAGAGAUUGGUUUGCCUGUUAAUGAAUUCAAACUAAUCUCUACACUGCUGCCCAAGAGC >hsa-mir-3130-1 MI0014147 (SEQ ID NO: 1599)CUUGUCAUGUCUUACCCAGUCUCCGGUGCAGCCUGUUGUCAAGGCUGCACCGGAGACUGGGUAAGACAUGACAAG >hsa-mir-3130-2 MI0014148 (SEQ ID NO: 1600)CUUGUCAUGUCUUACCCAGUCUCCGGUGCAGCCUUGACAACAGGCUGCACCGGAGACUGGGUAAGACAUGACAAG >hsa-mir-3131 MI0014151 (SEQ ID NO: 1601)GAGUCGAGGACUGGUGGAAGGGCCUUUCCCCUCAGACCAAGGCCCUGGCCCCAGCUUCUUCUC >hsa-mir-3132 MI0014152 (SEQ ID NO: 1602)GGUGGGAUGGGUAGAGAAGGAGCUCAGAGGACGGUGCGCCUUGUUUCCCUUGAGCCCUCCCUCUCUCAUCCCACC >hsa-mir-3133 MI0014153 (SEQ ID NO: 1603)CAGAAAUUGUAAAGAACUCUUAAAACCCAAUAGUAAAAAGACAACCUGUUGAGUUUUAAGAGUUCUUUAUAUAUUCUG >hsa-mir-3134 MI0014155 (SEQ ID NO: 1604)UGUAUCCAAUGUGUAGUCUUUUAUCCCUCACAUGGAGUAAAAUAUGAUGGAUAAAAGACUACAUAUUGGGUACA >hsa-mir-3135a MI0014156 (SEQ ID NO: 1605)UCACUUUGGUGCCUAGGCUGAGACUGCAGUGGUGCAAUCUCAGUUCACUGCAGCCUUGACCUCCUGGGCUCAGGUGA >hsa-mir-3135b MI0016809 (SEQ ID NO: 1606)UGCCCAGGCUGGAGCGAGUGCAGUGGUGCAGUCAGUCCUAGCUCACUGCAGCCUCGAACUCCUGGGCU >hsa-mir-3136 MI0014158 (SEQ ID NO: 1607)AAUAUGAAACUGACUGAAUAGGUAGGGUCAUUUUUCUGUGACUGCACAUGGCCCAACCUAUUCAGUUAGUUCCAUAUU >hsa-mir-3137 MI0014160 (SEQ ID NO: 1608)UACAGGUCUGUAGCCUGGGAGCAAUGGGGUGUAUGGUAUAGGGGUAGCCUCGUGCUCCUGGGCUACAAACCUGUA >hsa-mir-3138 MI0014161 (SEQ ID NO: 1609)CCCUCCUCGGCACUUCCCCCACCUCACUGCCCGGGUGCCCACAAGACUGUGGACAGUGAGGUAGAGGGAGUGCCGAGGAGGG >hsa-mir-3139 MI0014162 (SEQ ID NO: 1610)GGCUCAGAGUAGGAGCUCAACAGAUGCCUGUUGACUGAAUAAUAAACAGGUAUCGCAGGAGCUUUUGUUAUGUGCC >hsa-mir-3140 MI0014163 (SEQ ID NO: 1611)CCUCUUGAGGUACCUGAAUUACCAAAAGCUUUAUGUAUUCUGAAGUUAUUGAAAAUAAGAGCUUUUGGGAAUUCAGGUAGUUCAGGAGUG >hsa-mir-3141 MI0014165 (SEQ ID NO: 1612)UCACCCGGUGAGGGCGGGUGGAGGAGGAGGGUCCCCACCAUCAGCCUUCACUGGGACGGGA >hsa-mir-3142 MI0014166 (SEQ ID NO: 1613)UUCAGAAAGGCCUUUCUGAACCUUCAGAAAGGCUGCUGAAUCUUCAGAAAGGCCUUUCUGAACCUUCAGAAAGGCUGCUGAA >hsa-mir-3143 MI0014167 (SEQ ID NO: 1614)UAGAUAACAUUGUAAAGCGCUUCUUUCGCGGUUGGGCUGGAGCAACUCUUUACAAUGUUUCUA >hsa-mir-3144 MI0014169 (SEQ ID NO: 1615)AACUACACUUUAAGGGGACCAAAGAGAUAUAUAGAUAUCAGCUACCUAUAUACCUGUUCGGUCUCUUUAAAGUGUAGUU >hsa-mir-3145 MI0014170 (SEQ ID NO: 1616)UAUAUGAGUUCAACUCCAAACACUCAAAACUCAUUGUUGAAUGGAAUGAGAUAUUUUGAGUGUUUGGAAUUGAACUCGUAUA >hsa-mir-3146 MI0014172 (SEQ ID NO: 1617)GCUAAGUCCCUUCUUUCUAUCCUAGUAUAACUUGAAGAAUUCAAAUAGUCAUGCUAGGAUAGAAAGAAUGGGACUUGGC >hsa-mir-3147 MI0014173 (SEQ ID NO: 1618)GUCCGGGUUGGGCAGUGAGGAGGGUGUGACGCCGCGAAGUGCACCUCGCCCUUGUCCAACUCGGAC >hsa-mir-3148 MI0014175 (SEQ ID NO: 1619)GAGUUAAGAUGGAAAAAACUGGUGUGUGCUUAUUGAUGUAGCCAACAAGCAUACAUCAGUUUUUUCCAACUUAACUC >hsa-mir-3149 MI0014176 (SEQ ID NO: 1620)AUACAUACAUGUACACACACAUGUCAUCCACACACAUACAUAUAUAUAUGUUUGUAUGGAUAUGUGUGUGUAUGUGUGUGUAU >hsa-mir-3150a MI0014177 (SEQ ID NO: 1621)GGGAAGCAGGCCAACCUCGACGAUCUCCUCAGCACCUGAACGCCAAGGCUGGGGAGAUCCUCGAGGUUGGCCUGCUUUCC >hsa-mir-3150b MI0016426 (SEQ ID NO: 1622)GAGGGAAAGCAGGCCAACCUCGAGGAUCUCCCCAGCCUUGGCGUUCAGGUGCUGAGGAGAUCGUCGAGGUUGGCCUGCUUCCCCUC >hsa-mir-3151 MI0014178 (SEQ ID NO: 1623)GGGGUGAUGGGUGGGGCAAUGGGAUCAGGUGCCUCAAAGGGCAUCCCACCUGAUCCCACAGCCCACCUGUCACCCC >hsa-mir-3152 MI0014179 (SEQ ID NO: 1624)GUGCAGAGUUAUUGCCUCUGUUCUAACACAAGACUAGGCUUCCCUGUGUUAGAAUAGGGGCAAUAACUCUGCAC >hsa-mir-3153 MI0014180 (SEQ ID NO: 1625)GACAAAUUUUAAAUGUCCCUGUCCCCUUCCCCCCAAUUAAAGUAGAUUGGGGGAAAGCGAGUAGGGACAUUUAAAAUUUGUU >hsa-mir-3154 MI0014182 (SEQ ID NO: 1626)GGCCCCUCCUUCUCAGCCCCAGCUCCCGCUCACCCCUGCCACGUCAAAGGAGGCAGAAGGGGAGUUGGGAGCAGAGAGGGGACC >hsa-mir-3155a MI0014183 (SEQ ID NO: 1627)UCCGGGCAUCACCUCCCACUGCAGAGCCUGGGGAGCCGGACAGCUCCCUUCCCAGGCUCUGCAGUGGGAACUGAUGCCUGGA >hsa-mir-3155b MI0016839 (SEQ ID NO: 1628)CCACUGCAGAGCCUGGGAAGGGAGCUGUCCGGCUCCCCAGGCUCUGCAGUGGGAGU >hsa-mir-3156-1 MI0014184 (SEQ ID NO: 1629)GCAGAAGAAAGAUCUGGAAGUGGGAGACACUUUUACUAUAUAUAGUGGCUCCCACUUCCAGAUCUUUCUCUCUGU >hsa-mir-3156-2 MI0014230 (SEQ ID NO: 1630)UGCAGAAGAAAGAUCUGGAAGUGGGAGACACUUUCACUAUAUAUAGUGGCUCCCACUUCCAGAUCUUUCUCUCUGUA >hsa-mir-3156-3 MI0014242 (SEQ ID NO: 1631)UGCAGAAGAAAGAUCUGGAAGUGGGAGACACUUUCACUAUAUAUAGUGGCUCCCACUUCCUGAUCUUUCUCUCUGUA >hsa-mir-3157 MI0014185 (SEQ ID NO: 16323)GGGAAGGGCUUCAGCCAGGCUAGUGCAGUCUGCUUUGUGCCAACACUGGGGUGAUGACUGCCCUAGUCUAGCUGAAGCUUUUCCC >hsa-mir-3158-1 MI0014186 (SEQ ID NO: 1633)AUUCAGGCCGGUCCUGCAGAGAGGAAGCCCUUCUGCUUACAGGUAUUGGAAGGGCUUCCUCUCUGCAGGACCGGCCUGAAU >hsa-mir-3158-2 MI0014187 (SEQ ID NO: 1634)AUUCAGGCCGGUCCUGCAGAGAGGAAGCCCUUCCAAUACCUGUAAGCAGAAGGGCUUCCUCUCUGCAGGACCGGCCUGAAU >hsa-mir-3159 MI0014188 (SEQ ID NO: 1635)CCAAAGUCCUAGGAUUACAAGUGUCGGCCACGGGCUGGGCACAGUGGCUCACGCCUGUAAUCCCAGCAUUUUGG >hsa-mir-3160-1 MI0014189 (SEQ ID NO: 1636)GGACCUGCCCUGGGCUUUCUAGUCUCAGCUCUCCUCCAGCUCAGCUGGUCAGGAGAGCUGAGACUAGAAAGCCCAGGGCAGGUUC >hsa-mir-3160-2 MI0014190 (SEQ ID NO: 1637)ACCUGCCCUGGGCUUUCUAGUCUCAGCUCUCCUGACCAGCUGAGCUGGAGGAGAGCUGAGACUAGAAAGCCCAGGGCAGGU >hsa-mir-3161 MI0014191 (SEQ ID NO: 1638)CCUCGAGAGCUGAUAAGAACAGAGGCCCAGAUUGAAGUUGAAUAGUGCUGGGCCUUUGUUUUUACCAAGUUCCCUGG >hsa-mir-3162 MI0014192 (SEQ ID NO: 1639)CUGACUUUUUUAGGGAGUAGAAGGGUGGGGAGCAUGAACAAUGUUUCUCACUCCCUACCCCUCCACUCCCCAAAAAAGUCAG >hsa-mir-3163 MI0014193 (SEQ ID NO: 1640)UUCCUCAUCUAUAAAAUGAGGGCAGUAAGACCUUCCUUCCUUGUCUUACUACCCCCAUUUUAUAGAUGAGGAA >hsa-mir-3164 MI0014194 (SEQ ID NO: 1641)CUUGGAAACUGUGACUUUAAGGGAAAUGGCGCACAGCAGACCCUGCAAUCAUGCCGUUUUGCUUGAAGUCGCAGUUUCCCAGG >hsa-mir-3165 MI0014195 (SEQ ID NO: 1642)CCGGUGGCAAGGUGGAUGCAAUGUGACCUCAACUCUUGGUCCUCUGAGGUCACAUUGUAUCCACCUUACCACUGG >hsa-mir-3166 MI0014196 (SEQ ID NO: 1643)AAAUUUUUUUGAGGCCAGUAGGCAUUGUCUGCGUUAGGAUUUCUGUAUCAUCCUCCUAACGCAGACAAUGCCUACUGGCCUAAGAAAAAUUU >hsa-mir-3167 MI0014198 (SEQ ID NO: 1644)GGCUGUGGAGGCACCAGUAUUUCUGAAAUUCUUUUUUCUGAAAUUCUUCAGGAAGGAUUUCAGAAAUACUGGUGUCCCGACAGCC >hsa-mir-3168 MI0014199 (SEQ ID NO: 1645)AAGAUCAUGAGUUCUACAGUCAGACAGCCUGAGUUGGAGGCUCAUCUUCACUUCUUGCUGUGUGACCCUGGGCCAGUGACUU >hsa-mir-3169 MI0014200 (SEQ ID NO: 1646)AUGUGAAAACAUAGGACUGUGCUUGGCACAUAGCACAAAGUCUUAUGGUACUGUGUGCCAAGCAUAGUCCUGUGUUUUUACAU >hsa-mir-3170 MI0014201 (SEQ ID NO: 1647)CUGGUAACACUGGGGUUCUGAGACAGACAGUGUUAGCUCCAGAAGCAUUGCCUGUCUUAGAACCCCUAUGUUACCAG >hsa-mir-3171 MI0014202 (SEQ ID NO: 1648)UAUAUAUAGAGAUGUAUGGAAUCUGUAUAUAUCUAUAUAUAUGUGUAUAUAUAGAUUCCAUAAAUCUAUAUAUG >hsa-mir-3173 MI0014204 (SEQ ID NO: 1649)UCCCUGCCCUGCCUGUUUUCUCCUUUGUGAUUUUAUGAGAACAAAGGAGGAAAUAGGCAGGCCAGGGA >hsa-mir-3174 MI0014208 (SEQ ID NO: 1650)GUUACCUGGUAGUGAGUUAGAGAUGCAGAGCCCUGGGCUCCUCAGCAAACCUACUGGAUCUGCAUUUUAAUUCACAUGCAUGGUAAU >hsa-mir-3175 MI0014209 (SEQ ID NO: 1651)CCUGGGGGGCGGGGAGAGAACGCAGUGACGUCUGGCCGCGUGCGCAUGUCGGGCGCUUUCUCCUCCCCCUACCCAGG >hsa-mir-3176 MI0014210 (SEQ ID NO: 1652)UGGCCUCUCCAGUCUGCAGCUCCCGGCAGCCUCGGGCCACACUCCCGGGAUCCCCAGGGACUGGCCUGGGACUACCGGGGGUGGCGGCCG >hsa-mir-3177 MI0014211 (SEQ ID NO: 1653)CCACGUGCCAUGUGUACACACGUGCCAGGCGCUGUCUUGAGACAUUCGCGCAGUGCACGGCACUGGGGACACGUGGCACUGG >hsa-mir-3178 MI0014212 (SEQ ID NO: 1654)GAGGCUGGGCGGGGCGCGGCCGGAUCGGUCGAGAGCGUCCUGGCUGAUGACGGUCUCCCGUGCCCACGCCCCAAACGCAGUCUC >hsa-mir-3179-1 MI0014213 (SEQ ID NO: 1655)CAGGAUCACAGACGUUUAAAUUACACUCCUUCUGCUGUGCCUUACAGCAGUAGAAGGGGUGAAAUUUAAACGUCUGUGAUCCUG >hsa-mir-3179-2 MI0014216 (SEQ ID NO: 1656)CAGGAUCACAGACGUUUAAAUUACACUCCUUCUGCUGUGCCUUACAGCAGUAGAAGGGGUGAAAUUUAAACGUCUGUGAUCCUG >hsa-mir-3179-3 MI0014221 (SEQ ID NO: 1657)CAGGAUCACAGACGUUUAAAUUACACUCCUUCUGCUGUGCCUUACAGCAGUAGAAGGGGUGAAAUUUAAACGUCUGUGAUCCUG >hsa-mir-3180-1 MI0014214 (SEQ ID NO: 1658)CAGUGCGACGGGCGGAGCUUCCAGACGCUCCGCCCCACGUCGCAUGCGCCCCGGGAAAGCGUGGGGCGGAGCUUCCGGAGGCCCCGCCCUGCUG >hsa-mir-3180-2 MI0014215 (SEQ ID NO: 1659)GCGACGGGCGGAGCUUCCAGACGCUCCGCCCCACGUCGCAUGCGCCCCGGGAAAGCGUGGGGCGGAGCUUCCGGAGGCCCCGCCCUGC >hsa-mir-3180-3 MI0014217 (SEQ ID NO: 1660)CAGUGCGACGGGCGGAGCUUCCAGACGCUCCGCCCCACGUCGCAUGCGCCCCGGGAAAGCGUGGGGCGGAGCUUCCGGAGGCCCCGCCCUGCUG >hsa-mir-3180-4 MI0016408 (SEQ ID NO: 1661)GCUCCGCCCCACGUCGCAUGCGCCCCGGGAACGCGUGGGGCGGAGCUUCCGGAGGCCCCGCUCUGCUGCCGACCCUGUGGAGCGGAGGGUGAAGCCUCCGGAUGCCAGUCCCUCAUCGCUGGCCUGGUCGCGCUGUGGCGAAGGGGGCGGAGC >hsa-mir-3180-5 MI0016409 (SEQ ID NO: 1662)GCUCCGCCCCACGUCGCAUGCGCCCCGGGAACGCGUGGGGCGGAGCUUCCGGAGGCCCCGCCCUGCUGCCGACCCUGUGGAGCGGAGGGUGAAGCCUCCGGAUGCCAGUCCCUCAUCGCUGGCCCGGUCGCGCUGUGGCGAAGGGGGCGGAGC >hsa-mir-3181 MI0014223 (SEQ ID NO: 1663)CGGCGACCAUCGGGCCCUCGGCGCCGGCCCGUUAGUUGCCCGGGCCCGAGCCGGCCGGGCCCGCGGGUUGCCG >hsa-mir-3182 MI0014224 (SEQ ID NO: 1664)GCUGCUUCUGUAGUGUAGUCCGUGCAUCCGCCCUUCGAUGCUUGGGUUGGAUCAUAGAGCAGU >hsa-mir-3183 MI0014225 (SEQ ID NO: 1665)CUCUGCCCUGCCUCUCUCGGAGUCGCUCGGAGCAGUCACGUUGACGGAAUCCUCCGGCGCCUCCUCGAGGGAGGAGAGGCAGGG >hsa-mir-3184 MI0014226 (SEQ ID NO: 1666)AAGCAAGACUGAGGGGCCUCAGACCGAGCUUUUGGAAAAUAGAAAAGUCUCGCUCUCUGCCCCUCAGCCUAACUU >hsa-mir-3185 MI0014227 (SEQ ID NO: 1667)GAAUGGAAGAAGAAGGCGGUCGGUCUGCGGGAGCCAGGCCGCAGAGCCAUCCGCCUUCUGUCCAUGUC >hsa-mir-3186 MI0014229 (SEQ ID NO: 1668)AGCCUGCGGUUCCAACAGGCGUCUGUCUACGUGGCUUCAACCAAGUUCAAAGUCACGCGGAGAGAUGGCUUUGGAACCAGGGGCU >hsa-mir-3187 MI0014231 (SEQ ID NO: 1669)GCUGGCCCUGGGCAGCGUGUGGCUGAAGGUCACCAUGUUCUCCUUGGCCAUGGGGCUGCGCGGGGCCAGC >hsa-mir-3188 MI0014232 (SEQ ID NO: 1670)GGCGCCUCCUGCUCUGCUGUGCCGCCAGGGCCUCCCCUAGCGCGCCUUCUGGAGAGGCUUUGUGCGGAUACGGGGCUGGAGGCCU >hsa-mir-3189 MI0014233 (SEQ ID NO: 1671)GCCUCAGUUGCCCCAUCUGUGCCCUGGGUAGGAAUAUCCUGGAUCCCCUUGGGUCUGAUGGGGUAGCCGAUGC >hsa-mir-3190 MI0014235 (SEQ ID NO: 1672)CUGGGGUCACCUGUCUGGCCAGCUACGUCCCCACGGCCCUUGUCAGUGUGGAAGGUAGACGGCCAGAGAGGUGACCCCGG >hsa-mir-3191 MI0014236 (SEQ ID NO: 1673)GGGGUCACCUCUCUGGCCGUCUACCUUCCACACUGACAAGGGCCGUGGGGACGUAGCUGGCCAGACAGGUGACCCC >hsa-mir-3192 MI0014237 (SEQ ID NO: 1674)GGAAGGGAUUCUGGGAGGUUGUAGCAGUGGAAAAAGUUCUUUUCUUCCUCUGAUCGCCCUCUCAGCUCUUUCCUUCU >hsa-mir-3193 MI0014238 (SEQ ID NO: 1675)UCCUGCGUAGGAUCUGAGGAGUGGACGAGUCUCAUUACCCAGCUCCUGAGCAGGA >hsa-mir-3194 MI0014239 (SEQ ID NO: 1676)AGGUGGCAGGGCCAGCCACCAGGAGGGCUGCGUGCCACCCGGGCAGCUCUGCUGCUCACUGGCAGUGUCACCU >hsa-mir-3195 MI0014240 (SEQ ID NO: 1677)CCGCAGCCGCCGCGCCGGGCCCGGGUUGGCCGCUGACCCCCGCGGGGCCCCCGGCGGCCGGGGCGGGGGCGGGGGCUGCCCCGG >hsa-mir-3196 MI0014241 (SEQ ID NO: 1678)GGGUGGGGGCGGGGCGGCAGGGGCCUCCCCCAGUGCCAGGCCCCAUUCUGCUUCUCUCCCAGCU >hsa-mir-3197 MI0014245 (SEQ ID NO: 1679)GGCGAGGGGAGGCGCAGGCUCGGAAAGGCGCGCGAGGCUCCAGGCUCCUUCCCGAUCCACCGCUCUCCUCGCU >hsa-mir-3198-1 MI0014246 (SEQ ID NO: 1680)GACUGUGCUCUCACUGUUCACCCAGCACUAGCAGUACCAGACGGUUCUGUGGAGUCCUGGGGAAUGGAGAGAGCACAGUC >hsa-mir-3198-2 MI0017335 (SEQ ID NO: 1681)GACUCUGCUCUCACUGUUCACCCAGCACUAGCAGUACCAGAUGGUUCUGUGGAGUCCUGGGGAAUGGAGAGAGCACAGUC >hsa-mir-3199-1 MI0014247 (SEQ ID NO: 1682)GGUGACUCCAGGGACUGCCUUAGGAGAAAGUUUCUGGAAGUUCUGACAUUCCAGAAACUUUCUCCUAAGGCAGUCCCUGGGAGUCACU >hsa-mir-3199-2 MI0014248 (SEQ ID NO: 1683)GUGACUCCCAGGGACUGCCUUAGGAGAAAGUUUCUGGAAUGUCAGAACUUCCAGAAACUUUCUCCUAAGGCAGUCCCUGGAGUCAC >hsa-mir-3200 MI0014249 (SEQ ID NO: 1684)GGUGGUCGAGGGAAUCUGAGAAGGCGCACAAGGUUUGUGUCCAAUACAGUCCACACCUUGCGCUACUCAGGUCUGCUCGUGCCCU >hsa-mir-3201 MI0014250 (SEQ ID NO: 1685)GGGAUAUGAAGAAAAAUAAGAGGCUAGGAUUGCCUCUUAUUUUUACAUGCCC >hsa-mir-3202-1 MI0014252 (SEQ ID NO: 1686)UAUUAAUAUGGAAGGGAGAAGAGCUUUAAUGAUUGGAGUCAUUUUCAGAGCAUUAAAGCUCUUCUCCCUUCCAUAUUAAUG >hsa-mir-3202-2 MI0014253 (SEQ ID NO: 1687)AUUAAUAUGGAAGGGAGAAGAGCUUUAAUGCUCUGAAAAUGACUCCAAUCAUUAAAGCUCUUCUCCCUUCCAUAUUAAU >hsa-mir-3529 MI0017351 (SEQ ID NO: 1688)GGCACCAUUAGGUAGACUGGGAUUUGUUGUUGAGCGCAGUAAGACAACAACAAAAUCACUAGUCUUCCAGAUGGGGCC >hsa-mir-3545 MI0017343 (SEQ ID NO: 1689)GCGCCCGCCGGGUCUAGUGGUCCUAAACAUUUCACAAUUGCGCUACAGAACUGUUGAACUGUUAAGAACCACUGGACCCAGCGCGC >hsa-mir-3591 MI0017383 (SEQ ID NO: 1690)CAGUAGCUAUUUAGUGUGAUAAUGGCGUUUGAUAGUUUAGACACAAACACCAUUGUCACACUCCACAGCUCUG >hsa-mir-3605 MI0015995 (SEQ ID NO: 1691)ACUUUAUACGUGUAAUUGUGAUGAGGAUGGAUAGCAAGGAAGCCGCUCCCACCUGACCCUCACGGCCUCCGUGUUACCUGUCCUCUAGGUGGGACGCUCG >hsa-mir-3606 MI0015996 (SEQ ID NO: 1692)UUGUUGCUAUCUAGGUUAGUGAAGGCUAUUUUAAUUUUUUUAAAAUUUCUUUCACUACUUAGG >hsa-mir-3607 MI0015997 (SEQ ID NO: 1693)AAGGUUGCGGUGCAUGUGAUGAAGCAAAUCAGUAUGAAUGAAUUCAUGAUACUGUAAACGCUUUCUGAUGUACUACUCA >hsa-mir-3609 MI0015999 (SEQ ID NO: 1694)GUAACAGUAACUUUUAUUCUCAUUUUCCUUUUCUCUACCUUGUAGAGAAGCAAAGUGAUGAGUAAUACUGGCUGGAGCCC >hsa-mir-3610 MI0016000 (SEQ ID NO: 1695)AAGAGCCGCGGCGUAACGGCAGCCAUCUUGUUUGUUUGAGUGAAUCGGAAAGGAGGCGCCGGCUGUGGCGGCG >hsa-mir-3611 MI0016001 (SEQ ID NO: 1696)AGCAGGUCUAAUAAGAAUUUCUUUUUCUUCACAAUUAUGAAAGAAAAGAAAUUGUGAAGAAAGAAAUUCUUACUAGUUUUGCU >hsa-mir-3612 MI0016002 (SEQ ID NO: 1697)GGGACUGGGGAUGAGGAGGCAUCUUGAGAAAUGGAAGGAAUGGGAUCUACUUCCAGUUCACUAGAGGCGUCCUGACACCCCUAGCUC >hsa-mir-3613 MI0016003 (SEQ ID NO: 1698)UGGUUGGGUUUGGAUUGUUGUACUUUUUUUUUUGUUCGUUGCAUUUUUAGGAACAAAAAAAAAAGCCCAACCCUUCACACCACUUCA >hsa-mir-3614 MI0016004 (SEQ ID NO: 1699)GGUUCUGUCUUGGGCCACUUGGAUCUGAAGGCUGCCCCUUUGCUCUCUGGGGUAGCCUUCAGAUCUUGGUGUUUUGAAUUCUUACU >hsa-mir-3615 MI0016005 (SEQ ID NO: 1700)GACUCUGGGACGCUCAGACGCCGCGCGGGGCGGGGAUUGGUCUGUGGUCCUCUCUCGGCUCCUCGCGGCUCGCGGCGGCCGACGGUU >hsa-mir-3616 MI0016006 (SEQ ID NO: 1701)UGUCACUCCGCCAGCAUCAUGAAGUGCACUCAUGAUAUGUUUGCCCCAUCAGCGUGUCACGAGGGCAUUUCAUGAUGCAGGCGGGGUUGGCA >hsa-mir-3617 MI0016007 (SEQ ID NO: 1702)AGGUCAUAGAAAGACAUAGUUGCAAGAUGGGAUUAGAAACCAUAUGUCUCAUCAGCACCCUAUGUCCUUUCUCUGCCCU >hsa-mir-3618 MI0016008 (SEQ ID NO: 1703)UAAGCUGAGUGCAUUGUGAUUUCCAAUAAUUGAGGCAGUGGUUCUAAAAGCUGUCUACAUUAAUGAAAAGAGCAAUGUGGCCAGCUUG >hsa-mir-3619 MI0016009 (SEQ ID NO: 1704)ACGGCAUCUUUGCACUCAGCAGGCAGGCUGGUGCAGCCCGUGGUGGGGGACCAUCCUGCCUGCUGUGGGGUAAGGACGGCUGU >hsa-mir-3620 MI0016011 (SEQ ID NO: 1705)GUGAGGUGGGGGCCAGCAGGGAGUGGGCUGGGCUGGGCUGGGCCAAGGUACAAGGCCUCACCCUGCAUCCCGCACCCAG >hsa-mir-3621 MI0016012 (SEQ ID NO: 1706)GUGAGCUGCUGGGGACGCGGGUCGGGGUCUGCAGGGCGGUGCGGCAGCCGCCACCUGACGCCGCGCCUUUGUCUGUGUCCCACAG >hsa-mir-3622a MI0016013 (SEQ ID NO: 1707)AAUAGAGGGUGCACAGGCACGGGAGCUCAGGUGAGGCAGGGAGCUGAGCUCACCUGACCUCCCAUGCCUGUGCACCCUCUAUU >hsa-mir-3622b MI0016014 (SEQ ID NO: 1708)AGUGAUAUAAUAGAGGGUGCACAGGCAUGGGAGGUCAGGUGAGCUCAGCUCCCUGCCUCACCUGAGCUCCCGUGCCUGUGCACCCUCUAUUGGCU >hsa-mir-3646 MI0016046 (SEQ ID NO: 1709)UUCAGUAGGUUGGGUUCAUUUCAUUUUCAUGACAACCCUAUAUGGGAAAAUGUUGUGAAAAUGAAAUGAGCCCAGCCCAUUGAA >hsa-mir-3648 MI0016048 (SEQ ID NO: 1710)CGCGACUGCGGCGGCGGUGGUGGGGGGAGCCGCGGGGAUCGCCGAGGGCCGGUCGGCCGCCCCGGGUGCCGCGCGGUGCCGCCGGCGGCGGUGAGGCCCCGCGCGUGUGUCCCGGCUGCGGUCGGCCGCGCUCGAGGGGUCCCCGUGGCGUCCCCUUCCCCGCCGGCCGCCUUUCUCGCG >hsa-mir-3649 MI0016049 (SEQ ID NO: 1711)GCUUGGAACAGGCACCUGUGUGUGCCCAAGUGUUUCUAGCAAACACAGGGACCUGAGUGUCUAAGC >hsa-mir-3650 MI0016050 (SEQ ID NO: 1712)UCAAGGUGUGUCUGUAGAGUCCUGACUGCGUGCCAGGGGCUCUGUCUGGCACAUUUCUGA >hsa-mir-3651 MI0016051 (SEQ ID NO: 1713)GAUUCGAUGGGCCAUAGCAAUCCUGUGAUUUAUGCAUGGAGGCUGCUUCUCCUCAGCAGCUGCCAUAGCCCGGUCGCUGGUACAUGAUUC >hsa-mir-3652 MI0016052 (SEQ ID NO: 1714)CGGCUGGAGGUGUGAGGAUCCGAACCCAGGGGUGGGGGGUGGAGGCGGCUCCUGCGAUCGAAGGGGACUUGAGACUCACCGGCCGCACGCCAUGAGGGCCCUGUGGGUGCUGGGCCUCUGCUGCGUCCUGC >hsa-mir-3653 MI0016053 (SEQ ID NO: 1715)UCCCUGGGGACCCCUGGCAGCCCCUCCUGAUGAUUCUUCUUCCUGAGCACGCUCAUGAUGAGCAAACUGAGCCUCUAAGAAGUUGACUGAAGGGGCUGCUUCCCCAAGGA >hsa-mir-3654 MI0016054 (SEQ ID NO: 1716)UUCAUGAGCUGCAAUCUCAUCACUGGAAUGUUCCAGCGACUGGACAAGCUGAGGAA >hsa-mir-3655 MI0016055 (SEQ ID NO: 1717)GCUUGUCGCUGCGGUGUUGCUGUUGGAGACUCGAUUGUUGGUGACAGCGAAAGAACGAUAACAAAAUGCCGGAGCGAGAUAGU >hsa-mir-3656 MI0016056 (SEQ ID NO: 1718)CUUUCGGCCAGCGGGACGGCAUCCGAGGUGGGCUAGGCUCGGGCCCGUGGCGGGUGCGGGGGUGGGAGG >hsa-mir-3657 MI0016057 (SEQ ID NO: 1719)UGUGUCCCAUAAUUAAAUAAUGAAAUCUGAAAUCACCAAUAAUGGGACACUAAUGUGAUUAAUGUUGUUGUGUCCCAUUAUUGGUGAUUUCAGAUUUCAUAUAUGAUUAAGGACAUA >hsa-mir-3658 MI0016058 (SEQ ID NO: 1720)UAUUUAAGAAAACACCAUGGAGAUGAAAUGCCUUUGAUUUUUUUUUUCUUUUUGUA >hsa-mir-3659 MI0016060 (SEQ ID NO: 1721)UCUACAAGCAGAUACAAGGAUGCCCUUGUACACAACACACGUGCUGCUUGUAUAGACAUGAGUGUUGUCUACGAGGGCAUCCUUGUGUCUGUGUGUGUG >hsa-mir-3660 MI0016061 (SEQ ID NO: 1722)GAAAGAAGAACUGGACAAAAUUAAAAUGCUCUUCUGUCAUUGUAAUAGUUCAUAUGGGCACUGACAGGAGAGCAUUUUGACUUUGUCAAGUGUGUCUGCU >hsa-mir-3661 MI0016062 (SEQ ID NO: 1723)CACCUUCUCGCAGAGGCUCUUGACCUGGGACUCGGACAGCUGCUUGCACUCGUUCAGCUGCUCGAUCCACUGGUCCAGCUCCUUGGUGAACACCUU >hsa-mir-3662 MI0016063 (SEQ ID NO: 1724)UGUGUUUUCCUCAACGCUCACAGUUACACUUCUUACUCUCAAUCCAUUCAUAUUGAAAAUGAUGAGUAGUGACUGAUGAAGCACAAAUCAGCCAA >hsa-mir-3663 MI0016064 (SEQ ID NO: 1725)CCCGGGACCUUGGUCCAGGCGCUGGUCUGCGUGGUGCUCGGGUGGAUAAGUCUGAUCUGAGCACCACACAGGCCGGGCGCCGGGACCAAGGGGGCUC >hsa-mir-3664 MI0016065 (SEQ ID NO: 1726)CUGUAAACUUGAAGGUAGGGAACUCUGUCUUCACUCAUGAGUACCUUCCAACACGAGCUCUCAGGAGUAAAGACAGAGUUCCCUACCUUCAAUGUGGAU >hsa-mir-3665 MI0016066 (SEQ ID NO: 1727)GCGGGCGGCGGCGGCGGCAGCAGCAGCAGGUGCGGGGCGGCGGCCGCGCUGGCCGCUCGACUCCGCAGCUGCUCGUUCUGCUUCUCCAGCUUGCGCACCAGCUCC >hsa-mir-3666 MI0016067 (SEQ ID NO: 1728)AGUAAGGUCCGUCAGUUGUAAUGAGACCCAGUGCAAGUGUAGAUGCCGACUCCGUGGCAGAGUUCAGCGUUUCACACUGCCUGGUCUCUGUCACUCUAUUGAAUUAGAUUG >hsa-mir-3667 MI0016068 (SEQ ID NO: 1729)UGAGGAUGAAAGACCCAUUGAGGAGAAGGUUCUGCUGGCUGAGAACCUUCCUCUCCAUGGGUCUUUCAUCCUCA >hsa-mir-3668 MI0016069 (SEQ ID NO: 1730)AUAUAUGAAAUGUAGAGAUUGAUCAAAAUAGUUUCUAUCAAAAUAGUUUUGAUCAAUCUCUGCAAUUUUAUAUAU >hsa-mir-3669 MI0016070 (SEQ ID NO: 1731)AUAUAUAUAUACGGAAUAUAUAUACGGAAUAUAUAUAUACGGAAUAUAUAUAUACGGAAUAUGUAUACGGAAUAUAUAUAU >hsa-mir-3670-1 MI0016071 (SEQ ID NO: 1732)UCUAGACUGGUAUAGCUGCUUUUGGAGCCUCACCUGCUGAGAGCUCACAGCUGUCCUUCUCUAGA >hsa-mir-3670-2 MI0019112 (SEQ ID NO: 1733)UCUAGACUGGUAUAGCUGCUUUUGGAGCCUCACCUGCUGAGAGCUCACAGCUGUCCUUCUCUAGA >hsa-mir-3671 MI0016072 (SEQ ID NO: 1734)AUGUUAUUGCUGCUGCUGUCACAUUUACAUGAAAAUAAAAUGUAAAUUAUUUUAUUUCUAUCAAAUAAGGACUAGUCUGCAGUGAUAU >hsa-mir-3672 MI0016073 (SEQ ID NO: 1735)UCUUUGUGAUUACCAUGAGACUCAUGUAAAACAUCUUAGACUAUUACAAGAUGUUUUAUGAGUCUCAUGAUAAUCACAAAGA >hsa-mir-3673 MI0016074 (SEQ ID NO: 1736)AUAUAUAUAUAUGGAAUGUAUAUACGGAAUAUAUAUAUAUAUGGAAUGUAUAUACGGAAUAUAUAUAUAUAUGGAAUGUAUAUACGGAAUAUAUAUAUAUAU >hsa-mir-3674 MI0016075 (SEQ ID NO: 1737)ACAUCACUAUUGUAGAACCUAAGAUUGGCCGUUUGAGAUGUCCUUUCAAGUUUUUGCAUUUCUGAUGU >hsa-mir-3675 MI0016076 (SEQ ID NO: 1738)GGAUGAUAAGUUAUGGGGCUUCUGUAGAGAUUUCUAUGAGAACAUCUCUAAGGAACUCCCCCAAACUGAAUUC >hsa-mir-3676 MI0016077 (SEQ ID NO: 1739)UUGGUUAAAGCGCCUGUCUAGUAAACAGGAGAUCCUGGGUUCGAAUCCCAGCGGUGCCUCCGUGUUUCCCCCACGCUUUUGCCAA >hsa-mir-3677 MI0016078 (SEQ ID NO: 1740)GGCAGUGGCCAGAGCCCUGCAGUGCUGGGCAUGGGCUUCUCGUGGGCUCUGGCCACGGCC >hsa-mir-3678 MI0016079 (SEQ ID NO: 1741)GAAUCCGGUCCGUACAAACUCUGCUGUGUUGAAUGAUUGGUGAGUUUGUUUGCUCAUUGAUUGAAUCACUGCAGAGUUUGUACGGACCGGAUUC >hsa-mir-3679 MI0016080 (SEQ ID NO: 1742)CGUGGUGAGGAUAUGGCAGGGAAGGGGAGUUUCCCUCUAUUCCCUUCCCCCCAGUAAUCUUCAUCAUG >hsa-mir-3680-1 MI0016081 (SEQ ID NO: 1743)AAAUUUAAGGAGGGACUCACUCACAGGAUUGUGCAAAUGCAAAGUUGGCUUUUGCAUGACCCUGGGAGUAGGUGCCUCCUUAAAUUU >hsa-mir-3680-2 MI0019113 (SEQ ID NO: 1744)AAAUUUAAGGAGGGACUCACUCACAGGAUUGUGCAAAUGCAAAGUUGGCUUUUGCAUGACCCUGGGAGUAGGUGCCUCCUUAAAUUU >hsa-mir-3681 MI0016082 (SEQ ID NO: 1745)ACUUCCAGUAGUGGAUGAUGCACUCUGUGCAGGGCCAACUGUGCACACAGUGCUUCAUCCACUACUGGAAGU >hsa-mir-3682 MI0016083 (SEQ ID NO: 1746)UAAGUUAUAUAUGUCUACUUCUACCUGUGUUAUCAUAAUAAAGGUGUCAUGAUGAUACAGGUGGAGGUAGAAAUAUAUAACUUA >hsa-mir-3683 MI0016084 (SEQ ID NO: 1747)GGGUGUACACCCCCUGCGACAUUGGAAGUAGUAUCAUCUCUCCCUUGGAUGCUACGAACAAUAUCACAGAAGGUGUACACCC >hsa-mir-3684 MI0016085 (SEQ ID NO: 1748)AAUCUAAAGGACCUGUACUAGGUUUAACAUGUUGAGCAUUACUCAUGUUAGACCUAGUACACGUCCUUUAGAUU >hsa-mir-3685 MI0016086 (SEQ ID NO: 1749)GUACAUUUCCUACCCUACCUGAAGACUUGAGAUUAUAGUCUUUGGGGGGAUGGGCAAAGUAC >hsa-mir-3686 MI0016087 (SEQ ID NO: 1750)CUCACCUCAUUCAUUUACCUUCUCUUACAGAUCACUUUUCUGCACUGGACAGUGAUCUGUAAGAGAAAGUAAAUGAAAGAGGUGAG >hsa-mir-3687 MI0016088 (SEQ ID NO: 1751)CGCGCGUGCGCCCGAGCGCGGCCCGGUGGUCCCUCCCGGACAGGCGUUCGUGCGACGUGUG >hsa-mir-3688-1 MI0016089 (SEQ ID NO: 1752)UCUUCACUUUCAAGAGUGGCAAAGUCUUUCCAUAUGUAUGUAUGUAUGUCUGUUACACAUAUGGAAAGACUUUGCCACUCUUUAAAGUGAAGA >hsa-mir-3688-2 MI0017447 (SEQ ID NO: 1753)UCACUUUAAAGAGUGGCAAAGUCUUUCCAUAUGUGUAACAGACAUACAUACAUACAUAUGGAAAGACUUUGCCACUCUUGAAAGUGA >hsa-mir-3689a MI0016090 (SEQ ID NO: 1754)CCUGGGAGGUGUGAUAUCAUGGUUCCUGGGAGGUGUGAUCCUGUGCUUCCUGGGAGGUGUGAUAUCGUGGUUCCUGGG >hsa-mir-3689b MI0016411 (SEQ ID NO: 1755)GAUCCUGUGCUCCCUGGGGGGUCUGAUCCUGUGCUUCCUGGGAGGUGUGAUAUCAUGGUUCCUGGGAGGUGUGAUCCCGUGCUUCCUGGGAGGUGUGAUAUUGUGGUUCCUGGGAGGUGUGAUCCCGUGCUCCCUGGGAGGUGUGAUC >hsa-mir-3689c MI0016832 (SEQ ID NO: 1756)GGGAGGUGUGAUAUCGUGGUUCCUGGGAGGUGUGAUAUCGUGGUUCCUGGGAGGUGUGAUAUUGUGGUUCCU >hsa-mir-3689d-1 MI0016834 (SEQ ID NO: 1757)UGGGAGGUGUGAUCUCACACUCGCUGGGAGGUGUGCUAUCGUCUUCCCCGGGAGGUGUGAUCCUGUUCUUCCUG >hsa-mir-3689d-2 MI0016835 (SEQ ID NO: 1758)ACUGGGAGGUGUGAUCUCACACUCGCUGGGAGGUGUGCUAUCGUCUUCCCUGGGAGGUGUGAUCCUGUUCUUCCUGAGCG >hsa-mir-3689e MI0016836 (SEQ ID NO: 1759)GGGAGGUGUGAUAUCAUGGUUCCUGGGAGGUAUGAUAUCGUGGUUCCUGGGAGGUGUGAUCCCGUGCUCCCU >hsa-mir-3689f MI0016837 (SEQ ID NO: 1760)AGGUGUGAUAUCGUGCUUCCUGGGACGUGUGAUGCUGUGCUUCCUGGGAGGUGUGAUCCCACACUC >hsa-mir-3690 MI0016091 (SEQ ID NO: 1761)CCCAUCUCCACCUGGACCCAGCGUAGACAAAGAGGUGUUUCUACUCCAUAUCUACCUGGACCCAGUGUAGAUGGG >hsa-mir-3691 MI0016092 (SEQ ID NO: 1762)UUGAGGCACUGGGUAGUGGAUGAUGGAGACUCGGUACCCACUGCUGAGGGUGGGGACCAAGUCUGCGUCAUCCUCUCCUCAGUGCCUCAA >hsa-mir-3692 MI0016093 (SEQ ID NO: 1763)CCAUUCCUGCUGGUCAGGAGUGGAUACUGGAGCAAUAGAUACAGUUCCACACUGACACUGCAGAAGUGG >hsa-mir-3713 MI0016134 (SEQ ID NO: 1764)GGUAUCCGUUUGGGGAUGGUUUCACUAUCCCCAGAUGGAUACCAA >hsa-mir-3714 MI0016135 (SEQ ID NO: 1765)GAAGGCAGCAGUGCUCCCCUGUGACGUGCUCCAUCACCGGGCAGGGAAGACACCGCUGCCACCUC >hsa-mir-3907 MI0016410 (SEQ ID NO: 1766)GGGUUGGAAAGCUGUAGGUGUGGAGGGGCAUGGAUACGGGGGCCAUGAGGGUGGGGUCCAGGCUGGACCAGGCCUGCCCUGAGUCCCCCAGCAGGUGCUCCAGGCUGGCUCACACCCUCUGCCUCUCUCUCUUCCUUCCUGGCCCCAACCC >hsa-mir-3908 MI0016412 (SEQ ID NO: 1767)GCCUGAGCAAUGUAGGUAGACUGUUUCUAAAAAAAUAAAAAGUUAAAAAAAUUUAUGUUAACGUGUAAUGUGUUUACUAAUUUUUUUUUUUUUUUUUGGAGACAGAGUCUCCCUCUGUCGCCAGGC >hsa-mir-3909 MI0016413 (SEQ ID NO: 1768)GGUAUGCUGUUGCGCUGUCCUUCCUCUGGGGAGCAGGCUCCGGGGGACAGGGAAAAGCACACAAGGAACUUGUCCUCUAGGGCCUGCAGUCUCAUGGGAGAGUGACAUGCACCAGGACC >hsa-mir-3910-1 MI0016414 (SEQ ID NO: 1769)CUUUUGCUGUCAGUUUUUCUGUUGCUUGUCUUGGUUUUAUGCCUUUUAUAUCAAGGCACAUAAAAGGCAUAAAACCAAGACAAGCAACAAAAAAAGGAUUGAUCACAGAAG >hsa-mir-3910-2 MI0016431 (SEQ ID NO: 1770)UUUUUUUGUUGCUUGUCUUGGUUUUAUGCCUUUUAUGUGCCUUGAUAUAAAAGGCAUAAAACCAAGACAAGCAACAGAAAAA >hsa-mir-3911 MI0016415 (SEQ ID NO: 1771)GGGUGAGGAUGUGUGUGGAUCCUGGAGGAGGCAGAGAAGACAGUGAGCUUGCCAGUUCUGGUUUCCAACACUUCCUUUCCUGCGCUUCUCGAUUCCCAGAUCUGCACCC >hsa-mir-3912 MI0016416 (SEQ ID NO: 1772)AGAGAGGAAUGAACAGUUAAAUUAUAACAUGUCCAUAUUAUGGGUUAGUUGUGGACACAUACUAACGCAUAAUAUGGACAUGUUAUAAUUUAACUGUUCCUUUCU >hsa-mir-3913-1 MI0016417 (SEQ ID NO: 1773)UUGUUUAUAAUAAACUGAAAUAUUUGGGACUGAUCUUGAUGUCUGCCAAAACCUUGGCAGACAUCAAGAUCAGUCCCAAAUAUUUCAGUUUAUUAUAGACAG >hsa-mir-3913-2 MI0016418 (SEQ ID NO: 1774)UGUCUAUAAUAAACUGAAAUAUUUGGGACUGAUCUUGAUGUCUGCCAAGGUUUUGGCAGACAUCAAGAUCAGUCCCAAAUAUUUCAGUUUAUUAUAAACA >hsa-mir-3914-1 MI0016419 (SEQ ID NO: 1775)UGGACUUCAGAUUUAACUUCUCAUUUUCUGGUUCCUUCUAAUGAGUAUGCUUAACUUGGUAGAAGGAACCAGAAAAUGAGAAGUUGAGUAGGAACUCUA >hsa-mir-3914-2 MI0016421 (SEQ ID NO: 1776)GAGUUCCUACUCAACUUCUCAUUUUCUGGUUCCUUCUACCAAGUUAAGCAUACUCAUUAGAAGGAACCAGAAAAUGAGAAGUUAAAUCUGAAGUC >hsa-mir-3915 MI0016420 (SEQ ID NO: 1777)CAAGUUGGCACUGUAGAAUAUUGAGGAAAAGAUGGUCUUAUUGCAAAGAUUUUCAAUAAGACCAUCCUUUCCUCAAUAUUCUGUGGUGUCAUCUUUG >hsa-mir-3916 MI0016422 (SEQ ID NO: 1778)AUCCCAGAGAAGAAGGAAGAAGAGGAAGAAAUGGCUGGUUCUCAGGUGAAUGUGUCUGGGUUCAGGGGAUGUGUCUCCUCUUUUCUUCUGGGAU >hsa-mir-3917 MI0016423 (SEQ ID NO: 1779)GGCGCUUUUGUGCGCGCCCGGGUCUGUUGGUGCUCAGAGUGUGGUCAGGCGGCUCGGACUGAGCAGGUGGGUGCGGGGCUCGGAGGAGGCGGC >hsa-mir-3918 MI0016424 (SEQ ID NO: 1780)AGGCGGUUAAGCCAUGGGACAGGGCCGCAGAUGGAGACUGCUCAAGGUCAAAGGGGUCUCCAGCUGGGACCCUGCACCUGGUUCGUAGCCCCU >hsa-mir-3919 MI0016425 (SEQ ID NO: 1781)CCUGAGCACCAUUUACUGAGUCCUUUGUUCUCUACUAGUUUGUAGUAGUUCGUAGCAGAGAACAAAGGACUCAGUAAAUGGUGCUCAGG >hsa-mir-3920 MI0016427 (SEQ ID NO: 1782)ACUGAGUGAGGGAGUCAGAGAGUUAAGAGAAUUAGUACAGGUGAGAUUGUACUGAUUAUCUUAACUCUCUGACCCCCUCACUCAGU >hsa-mir-3921 MI0016428 (SEQ ID NO: 1783)CCUAGCCCAGUACAAGGCAUAUGGUACUCAAGAGACUUAGAAAUCCCUAAGUCUCUGAGUACCAUAUGCCUUGUACUGGGCUAGG >hsa-mir-3922 MI0016429 (SEQ ID NO: 1784)GGAAGAGUCAAGUCAAGGCCAGAGGUCCCACAGCAGGGCUGGAAAGCACACCUGUGGGACUUCUGGCCUUGACUUGACUCUUUC >hsa-mir-3923 MI0016430 (SEQ ID NO: 1785)GGUAGAGUGAGCUCUAAUCCAAUAUUACUAGCUUCUUUAUAAGAAGAGGAAACUAGUAAUGUUGGAUUAGGGCUCACUCUACU >hsa-mir-3924 MI0016432 (SEQ ID NO: 1786)UAAAUGAAAAAGUAGUAGUCAAAUAUGCAGAUCUAUGUCAUAUAUACAGAUAUGUAUAUGUGACUGCUACUUUUUUGUUUA >hsa-mir-3925 MI0016433 (SEQ ID NO: 1787)GUGGGAAUAGCAAGAGAACUGAAAGUGGAGCCUGUCACAUCUCCAGACUCCAGUUUUAGUUCUCUUGCUAUUUCCAC >hsa-mir-3926-1 MI0016434 (SEQ ID NO: 1788)AAAAUGGAGCUGGCCAAAAAGCAGGCAGAGACUUUAAAAGCGUCUCUGCCUGCUUUUUGGCCAGCUCCGUUUU >hsa-mir-3926-2 MI0016437 (SEQ ID NO: 1789)GGAGCUGGCCAAAAAGCAGGCAGAGACGCUUUUAAAGUCUCUGCCUGCUUUUUGGCCAGCUCC >hsa-mir-3927 MI0016435 (SEQ ID NO: 1790)UGCCAAUGCCUAUCACAUAUCUGCCUGUCCUAUGACAAACAUGGCAGGUAGAUAUUUGAUAGGCAUUGGCA >hsa-mir-3928 MI0016438 (SEQ ID NO: 1791)GCUGAAGCUCUAAGGUUCCGCCUGCGGGCAGGAAGCGGAGGAACCUUGGAGCUUCGGC >hsa-mir-3929 MI0016439 (SEQ ID NO: 1792)AGUGGCUCACACCAGUAAUCCCAGCACUUUGGGAGGCUGAUGUGAGUAGACCACU >hsa-mir-3934 MI0016590 (SEQ ID NO: 1793)CACAGCCCUUCCUGUCCCCAGUUUUCAGGUGUGGAAACUGAGGCAGGAGGCAGUGAAGUAACUUGCUCAGGUUGCACAGCUGGGAAGUGGAGCAGGGAUUUGAAUCC >hsa-mir-3935 MI0016591 (SEQ ID NO: 1794)GGAUGUGUUCCUGUCCCAGAAGGAGCUGAUGGUUGUAUCUAUGAAGGUAAGCAUUUUUGUAGAUACGAGCACCAGCCACCCUAAGCAAAGGCAGAGAAUGCUUA >hsa-mir-3936 MI0016592 (SEQ ID NO: 1795)AUGAUUCAGAGCAUCUGUCCAGUGUCUGCUGUAGAUCCCUCAAAUCCGUGUUUGGACGCUUCUGGUAAGGGGUGUAUGGCAGAUGCACCCGACAGAUGCACUUGGCAGCA >hsa-mir-3937 MI0016593 (SEQ ID NO: 1796)AGAAGAAUGCCCAACCAGCCCUCAGUUGCUACAGUUCCCUGUUGUUUCAGCUCGACAACAACAGGCGGCUGUAGCAAUGGGGGGCUGGAUGGGCAUCUCAAUGUGC >hsa-mir-3938 MI0016594 (SEQ ID NO: 1797)AGGAAUUUUUAACCCGAUCACUAGAUUAUCUACAAGGGAAUUUUUUUUUAAUUUAAAAAAUUCCCUUGUAGAUAACCCGGUGGUCAGGUUGGAUGGCUCCAUG >hsa-mir-3939 MI0016596 (SEQ ID NO: 1798)CUGGCUUCCAAAGGCCUCUGUGUGUUCCUGUAUGUGGGCGUGCACGUACCUGUCACAUGUGUACGCGCAGACCACAGGAUGUCCACACUGGCUUCCAAACACAUCU >hsa-mir-3940 MI0016597 (SEQ ID NO: 1799)GCUUAUCGAGGAAAAGAUCGAGGUGGGUUGGGGCGGGCUCUGGGGAUUUGGUCUCACAGCCCGGAUCCCAGCCCACUUACCUUGGUUACUCUCCUUCCUUCU >hsa-mir-3941 MI0016598 (SEQ ID NO: 1800)GAGUCAGAAUUCUCAUCAGGCUGUGAUGCUCAGUUGUGUGUAGAUUGAAAGCCCUAAUUUUACACACAACUGAGGAUCAUAGCCUGAUGGUUCCUUUUUGUUU >hsa-mir-3942 MI0016599 (SEQ ID NO: 1801)UCUUCAGUAUGACACCUCAAAGAAGCAAUACUGUUACCUGAAAUAGGCUGCGAAGAUAACAGUAUUUCAGAUAACAGUAUUACAUCUUUGAAGUGUCAUAUUCACUGAC >hsa-mir-3943 MI0016600 (SEQ ID NO: 1802)CACACAGACGGCAGCUGCGGCCUAGCCCCCAGGCUUCACUUGGCGUGGACAACUUGCUAAGUAAAGUGGGGGGUGGGCCACGGCUGGCUCCUACCUGGAC >hsa-mir-3944 MI0016601 (SEQ ID NO: 1803)UCCACCCAGCAGGCGCAGGUCCUGUGCAGCAGGCCAACCGAGAAGCGCCUGCGUCUCCCAUUUUCGGGCUGGCCUGCUGCUCCGGACCUGUGCCUGAUCUUAAUGCUG >hsa-mir-3945 MI0016602 (SEQ ID NO: 1804)GAUGUUGAUGCACGUGACGGGGAGGGCAUAGGAGAGGGUUGAUAUAAAAUGCAAUUACAGCCUCUUAUGCUUUCCAAAGUGGGGGAUGAUUCAAUGAU >hsa-mir-3960 MI0016964 (SEQ ID NO: 1805)GGCGCCCCGGCUCCCCGCGCCCCCGAUCGGGGCCGCCGCUAGUAGUGGCGGCGGCGGAGGCGGGGGCAGCGGCGGCGGCGGCGGAGGCGCC >hsa-mir-3972 MI0016990 (SEQ ID NO: 1806)GCCCAUUUGCCUUGGCUUGGGGUGGCAGUCCUGUGGGAAUGAGAGAUGCCAAACUGGACCUGCCAGCCCCGUUCCAGGGCACAGCAU >hsa-mir-3973 MI0016991 (SEQ ID NO: 1807)GCCCAGGGUAGCUCUCUGUAUUGCUUGUUACAUUUUAGGAUUGCUUGCCCCUUGCUCCAAUGGUGCAGGCAGAAGAAAUGCAAACAAAGUACAGCAUUAGCCUUAGC >hsa-mir-3974 MI0016992 (SEQ ID NO: 1808)GUUCAGGGAAAAGGUCAUUGUAAGGUUAAUGCACCCAUAUUUUAAUAUCAAACUAUGACAAAUUUGACUACAGCCUUUCCGUACCCCUGCCAAAAC >hsa-mir-3975 MI0016993 (SEQ ID NO: 1809)CAUGUAGGUGAGUGAUUGCUAUUUCAAAAGACUAGAGGUAGGAAAUGAGGCUAAUGCACUACUUCACAUG >hsa-mir-3976 MI0016994 (SEQ ID NO: 1810)UGAGGGAUAUAGAGAGCAGGAAGAUUAAUGUCAUAUUGGAGUUGGACUGCAGGGCUUCCUUUACACAAUAAAUAUUGUAUGAAGUGCUGAUGUAACCUUUACUGCAGCAUGACAUGGGAUUUGGCUGUUUUUAUGGCUC >hsa-mir-3977 MI0016995 (SEQ ID NO: 1811)UUGUGCUUCAUCGUAAUUAACCUUAAGUGGUUCGGGUAAAUCACUUUAAUUUGUUAUGUGUUGGCAGAAU >hsa-mir-3978 MI0016996 (SEQ ID NO: 1812)UCAGUGGAAAGCAUGCAUCCAGGGUGUGGAGCCAAAAUUAGAAGGGCCAAAAUUCUACCUGGCCCACUACCACAGCAACCUUGGGCAUCGUUUUCUUUUGA >hsa-mir-4251 MI0015861 (SEQ ID NO: 1813)CACGUCCUCCAGCUUUUUUCCUUAGUGGCCAAUUCCUGAGAAAAGGGCCAACGUGCUUCCA >hsa-mir-4252 MI0015864 (SEQ ID NO: 1814)UGGGGGGCUGGCAGCUCAUCAGUCCAGGCCAUCUGGCCACUGAGUCAGCACCAGCGCCCAAUC >hsa-mir-4253 MI0015860 (SEQ ID NO: 1815)CCAGCCAUCGCCCUUGAGGGGCCCUAGGACUUACUUGUGCAGGGCAUGUCCAGGGGGUCCAGGUCUGC >hsa-mir-4254 MI0015862 (SEQ ID NO: 1816)CUUGGGAGGAGGGUGGGGUGGCUCCUCUGCAGUGAGUAGGUCUGCCUGGAGCUACUCCACCAUCUCCCCCAGCCCC >hsa-mir-4255 MI0015863 (SEQ ID NO: 1817)GAGCAUCCUUCAGUGUUCAGAGAUGGAGUCAGUAUUGGUCUGGCCAUUUUUAGGGCAAAGAGGCAGCAUCAU >hsa-mir-4256 MI0015855 (SEQ ID NO: 1818)UGUUCCAUUUAUCUGACCUGAUGAAGGUCUCCUGGCAUUGAUUAGGUCUGAUGAUCCAUUUCUG >hsa-mir-4257 MI0015856 (SEQ ID NO: 1819)GGCUUAGAAACAGUCCCUAGGUAGGAUUUGGGGAGGAGCUAAGAAGCCCCUACAGGGCCCAGAGGUGGGGACUGAGCCUUAGUUGG >hsa-mir-4258 MI0015857 (SEQ ID NO: 1820)ACGCCCCCCGCCCCGCCACCGCCUUGGAGGCUGACCUCUUACUUUCGGUCGGUCUUCUUCCCUGGGCUUGGUUUGGGGGCGGGGGAGUGUC >hsa-mir-4259 MI0015858 (SEQ ID NO: 1821)GAUGGGCCCCUUGUGUCCUGAAUUGGGUGGGGGCUCUGAGUGGGGAAAGUGGGGGCCUAGGGGAGGUCACAGUUGGGUCUAGGGGUCAGGAGGGCCCAGGA >hsa-mir-4260 MI0015859 (SEQ ID NO: 1822)AACAAGGUGACUUGGGGCAUGGAGUCCCACUUCCUGGAGCCCACACCCCAGCUUGUCACACACCAAC >hsa-mir-4261 MI0015868 (SEQ ID NO: 1823)GGUGGAAGUGGGUUCCUCCCAGUUCCUGAGACAGGAAACAGGGACCCAGGAGACCAGC >hsa-mir-4262 MI0015872 (SEQ ID NO: 1824)GAAAGCUGCAGGUGCUGAUGUUGGGGGGACAUUCAGACUACCUGCAGCAGAGCC >hsa-mir-4263 MI0015876 (SEQ ID NO: 1825)AUAGUGCUCUUCAGGGUUUUACUUGGGAGAUUGGAGUGGCCAGUGUUCCUAAACAAUUCUAAGUGCCUUGGCCCACAACAUAC >hsa-mir-4264 MI0015877 (SEQ ID NO: 1826)AAAGCUGGAUACUCAGUCAUGGUCAUUGUAACAUGAUAGUGACAGGUACUGGGUAAGACUGCAUAG >hsa-mir-4265 MI0015869 (SEQ ID NO: 1827)UGCAGUGGGUUGGAGCUUCAGCCUACACCUGUAAAGAAUUGGUCAGCCUGGGGACUGGUGAUCUCUGCAGCUGUGGGCUCAGCUCUGGGCUGGGCCUGG >hsa-mir-4266 MI0015870 (SEQ ID NO: 1828)CCACUGCUGGCCGGGGCCCCUACUCAAGGCUAGGAGGCCUUGGCCAAGGACAGUC >hsa-mir-4267 MI0015871 (SEQ ID NO: 1829)CUCAGCAGGCUCCAGCUCGGUGGCACUGGGGGAAGGCUCCAGACCCCAGCCUCUGUCAUCCCUGCAUGGAGCCCACAUCUCC >hsa-mir-4268 MI0015874 (SEQ ID NO: 1830)AUGCACAUCAGGUUCUAGAGGUUUUGCCCUAGCGGCUCCUCCUCUCAGGAUGUGAUGUCACCUG >hsa-mir-4269 MI0015875 (SEQ ID NO: 1831)ACAGCGCCCUGCAGGCACAGACAGCCCUGGCUUCUGCCUCUUUCUUUGUGGAAGCCACUCUGUCAGGCCUGGGAUGGAGGGGCA >hsa-mir-4270 MI0015878 (SEQ ID NO: 1832)ACAAAUAGCUUCAGGGAGUCAGGGGAGGGCAGAAAUAGAUGGCCUUCCCCUGCUGGGAAGAAAGUGGGUC >hsa-mir-4271 MI0015879 (SEQ ID NO: 1833)AAAUCUCUCUCCAUAUCUUUCCUGCAGCCCCCAGGUGGGGGGGAAGAAAAGGUGGGGAAUUAGAUUC >hsa-mir-4272 MI0015880 (SEQ ID NO: 1834)UUUUCUGCACAAAUUAAUCAGUUAAUGCAUAGAAAGCAUUCAACUAGUGAUUGUGUUAUAAGAG >hsa-mir-4273 MI0015881 (SEQ ID NO: 1835)UCCCCUGUGUGUGUUCUCUGAUGGACAGUAAGCCUUGACUUAUGGCUAAAUGCUUCUUCACAAUGGUCACAUGCAUAGGGCUUU >hsa-mir-4274 MI0015884 (SEQ ID NO: 1836)GGGGCAUUUAGGGUAACUGAGCUGCUGCCGGGGCCUGGCGCUCCUCUACCUUGUCAGGUGACCCAGCAGUCCCUCCCCCUGCAUGGUGCCC >hsa-mir-4275 MI0015883 (SEQ ID NO: 1837)ACAUUUUUGUCCAAUUACCACUUCUUUUUGCCACCUGAGCACAGUCAGCAGUCAGCAUAAAAAAGUGAUAAUGGGAAGUUAAUGUCU >hsa-mir-4276 MI0015882 (SEQ ID NO: 1838)CACAGUCUGACUCAGUGACUCAUGUGCUGGCAGUGGCCACGUAAAUAGAGCUACUGUGUCUGAAAGCAAU >hsa-mir-4277 MI0015886 (SEQ ID NO: 1839)CUGGGUCGAGGCAGUUCUGAGCACAGUACACUGGGCUGCCCCCACUGCCCAGUGCCCUGCUCAGCUCAAGUCCUUGUGCCCCUC >hsa-mir-4278 MI0015888 (SEQ ID NO: 1840)AUCUAACACCAGGAGAAUCCCAUAGAACAUUGACAUCAACACUAGGGGGUUUGCCCUUGUGGGGAAGAA >hsa-mir-4279 MI0015887 (SEQ ID NO: 1841)UGCUCUGUGGAGCUGAGGAGCAGAUUCUCUCUCUCUCCUCCCGGCUUCACCUCCUGAG >hsa-mir-4280 MI0015889 (SEQ ID NO: 1842)AAUCAGGGUGGAGUGUAGUUCUGAGCAGAGCCUUAAAGGAUGAGGUAUGUUCAAGACUGAAUGACACCUUUGUGAU >hsa-mir-4281 MI0015885 (SEQ ID NO: 1843)GCUGGGGGUCCCCCGACAGUGUGGAGCUGGGGCCGGGUCCCGGGGAGGGGGGUUCUGGGCAG >hsa-mir-4282 MI0015890 (SEQ ID NO: 1844)GGUGAAGUUCCAGGGGAAGAUUUUAGUAUGCCACAUUUCUAAAAUUUGCAUCCAGGAACAUCAUCCU >hsa-mir-4283-1 MI0015892 (SEQ ID NO: 1845)ACUCUGAUCCUGGGGCUCAGCGAGUUUGCAAGGGGUGUUUCUGUCCAUGGUCAGGCUUGCCAGCCUUGGUCCUUGGGCCC >hsa-mir-4283-2 MI0015982 (SEQ ID NO: 1846)ACUCUGAUCCUGGGGCUCAGCGAGUUUGCAAGGGGUGUUUCUGUCCAUGGUCAGGCUUGCCAGCCUUGGUCCUUGGGCCC >hsa-mir-4284 MI0015893 (SEQ ID NO: 1847)GUUCUGUGAGGGGCUCACAUCACCCCAUCAAAGUGGGGACUCAUGGGGAGAGGGGGUAGUUAGGAGCUUUGAUAGAGGCGG >hsa-mir-4285 MI0015891 (SEQ ID NO: 1848)AUUAGCUGGGGCGGCGAGUCCGACUCAUCAAUAUUUUAAGGAAUGACCCGGCCUUGGGGUGCGGAAUUGCUGCGCGGGCGGGGGC >hsa-mir-4286 MI0015894 (SEQ ID NO: 1849)UACUUAUGGCACCCCACUCCUGGUACCAUAGUCAUAAGUUAGGAGAUGUUAGAGCUGUGAGUACCAUGACUUAAGUGUGGUGGCUUAAACAUG >hsa-mir-4287 MI0015895 (SEQ ID NO: 1850)UAGUUCUUUUUCUCCCUUGAGGGCACUUUUCAGUUCCUGAGAUCAAUGUGGUCCCUACUGGGGAGACCAUAGGAGCCC >hsa-mir-4288 MI0015896 (SEQ ID NO: 1851)AUGGAGGUGGAGAGUCAUCAGCAGCACUGAGCAGGCAGUGUUGUCUGCUGAGUUUCCACGUCAUUUG >hsa-mir-4289 MI0015898 (SEQ ID NO: 1852)CCUUGGGAGGGCAUUGUGCAGGGCUAUCAGGCAGUUUCCUGGGCCCUGUCUGCAGAGCCUAAACAGAUCA >hsa-mir-4290 MI0015899 (SEQ ID NO: 1853)GCCACCAAGAAGGUGAAGGGAGGGUCAGUCCCAAUCUGAAUCCCACCAAAAUAGGUGGUAGAGGGUUGCCCUCCUUUCUUCCCUCACCUCUGACC >hsa-mir-4291 MI0015900 (SEQ ID NO: 1854)CGCCGGGGGCUUCAGCAGGAACAGCUGGGUGGAGGCAGAGCUGUUCUGCUGUGGCUGCAGCCCUG >hsa-mir-4292 MI0015897 (SEQ ID NO: 1855)GAGACACCAGAAGGCCACCUGCUUAGGAGGCCAGAGGUGCCCCUGGGCCGGCCUUGGUGAGGGGCCC >hsa-mir-4293 MI0015826 (SEQ ID NO: 1856)AGAGACACCUGUUCCUUGGGAAGCUGGUGACAUUGCUAAUUCAUUUCACACCAGCCUGACAGGAACAGCCUGACUGAA >hsa-mir-4294 MI0015827 (SEQ ID NO: 1857)CCGAUGCCUCGGGAGUCUACAGCAGGGCCAUGUCUGUGAGGGCCCAAGGGUGCAUGUGUCUCCCAGGUUUCGGUGC >hsa-mir-4295 MI0015822 (SEQ ID NO: 1858)CUUUGUGGAACAGUGCAAUGUUUUCCUUGCCUGUGGCAAGACCACUUCGGUUCAAGGCUAAGAAACUAGACUGUUCCUACAGAGA >hsa-mir-4296 MI0015823 (SEQ ID NO: 1859)UUGGGCUUUGAUGUGGGCUCAGGCUCAGAGGGCUGAAGUGGUUGUGGGGAGGGGCUUCUGGGGACUGUGUCCAUGUCUCUGUCGUUUU >hsa-mir-4297 MI0015824 (SEQ ID NO: 1860)AGCACGCACGUGCCUUCCUGUCUGUGCCUGCCUUCGAAGUGCACGGCAGGGCCAGGACGGGUCGCUGUGGGUGGGG >hsa-mir-4298 MI0015830 (SEQ ID NO: 1861)GGGGAGGUACCUGGGACAGGAGGAGGAGGCAGCCUUGCCUCAGAAACCAAACUGUCAAAAGUGUAGGUUCCAC >hsa-mir-4299 MI0015829 (SEQ ID NO: 1862)GGGUUCUGACCAAUCAUGUUACAGUGUUUUCUCCUUUAGAGAGAGCUGGUGACAUGAGAGGCAGAAAAAGGA >hsa-mir-4300 MI0015831 (SEQ ID NO: 1863)UGAGUUUAGAAGAGGGCCAGCUAAAUCAGCAGAGACAUGAGGUGAUCAAAAACCUUUUUUCAAAGCAGUGGGAGCUGGACUACUUCUGAACCAAUA >hsa-mir-4301 MI0015828 (SEQ ID NO: 1864)ACCAGCCACCUCCCACUACUUCACUUGUGAACAUUGCAUUCGUGGAGGGUGGCAGGUGCAGCUCUG >hsa-mir-4302 MI0015833 (SEQ ID NO: 1865)UCAGGAGGGACCAGUGUGGCUCAGCGAGGUGGCUGAGUUUACUUAAGGUAUUGGAAUGAG >hsa-mir-4303 MI0015834 (SEQ ID NO: 1866)AGAAAAUAGCUUCUGAGCUGAGGACAGCUUGCUCUGCUUUUCUUUAGCUUAGGAGCUAACCAUGGU >hsa-mir-4304 MI0015832 (SEQ ID NO: 1867)AGAGAAGUGGCCGGCAUGUCCAGGGCAUCCCCAUUGCUCUGUGACUGCUGCCAUCCUUCUCC >hsa-mir-4305 MI0015835 (SEQ ID NO: 1868)CUGCCUUAGACCUAGACACCUCCAGUUCUGGGUUCUUAGAGGCCUAAUCCUCUACAAACUCAGUUUUCAGACUGUGAGGGAAAUUCUCUGUCUUAUUGCUUU >hsa-mir-4306 MI0015836 (SEQ ID NO: 1869)AAGCUGCUUAGUGUCCUUAGAGUCUCCAGAGGCAUCCCUAACCCAGAAUCUUUUGACUGUCCUCUGGAGAGAAAGGCAGUAGGUCUGUACC >hsa-mir-4307 MI0015838 (SEQ ID NO: 1870)UCAGAAGAAAAAACAGGAGAUAAAGUUUGUGAUAAUGUUUGUCUAUAUAGUUAUGAAUGUUUUUUCCUGUUUCCUUCAGGGCCA >hsa-mir-4308 MI0015839 (SEQ ID NO: 1871)UAUGGGUUCAGAGGGAACUCCAUUGGACAGAAAUUUCCUUUUGAGGAAAUCUUUCCCUGGAGUUUCUUCUUACCUUUUUCC >hsa-mir-4309 MI0015837 (SEQ ID NO: 1872)UCUGGGGGUUCUGGAGUCUAGGAUUCCAGGAUCUGGGUUUUGAGGUCUUGGGUUGUAGGGUCUGCGGUUUGAAGCCCCUCUUG >hsa-mir-4310 MI0015840 (SEQ ID NO: 1873)UGGCGUCUGGGGCCUGAGGCUGCAGAACAUUGCAGCAUUCAUGUCCCACCCCCACCA >hsa-mir-4311 MI0015841 (SEQ ID NO: 1874)UCAGAGAGGGGAAAGAGAGCUGAGUGUGACCUGGAGCAGCUCAGGAGGGCUUCCUGGGUGAGGUGGCAGGUUACAGGUUCGAUCUUUGGCCCUCAGAUUC >hsa-mir-4312 MI0015842 (SEQ ID NO: 1875)GAAAGGUUGGGGGCACAGAGAGCAAGGAGCCUUCCCCAGAGGAGUCAGGCCUUGUUCCUGUCCCCAUUCCUCAGAG >hsa-mir-4313 MI0015843 (SEQ ID NO: 1876)GAUCAGGCCCAGCCCCCUGGCCCCAAACCCUGCAGCCCCAGCUGGAGGAUGAGGAGAUGCUGGGCUUGGGUGGGGGAAUCAGGGGUGUAAAGGGGCCUGCU >hsa-mir-4314 MI0015846 (SEQ ID NO: 1877)GGCCAUUCCUCUCUGGGAAAUGGGACAGGUAGUGGCCACAGUGAGAAAGCUGGCCUGUCCUUCUGCCCCAGGGCCCAGAGUCUGUGACUGGA >hsa-mir-4315-1 MI0015844 (SEQ ID NO: 1878)UGGGCUUUGCCCGCUUUCUGAGCUGGACCCUCUCUCUACCUCUGGUGCAGAACUACAGCGGAAGGAAUCUCUG >hsa-mir-4315-2 MI0015983 (SEQ ID NO: 1879)UGGGCUUUGCCCGCUUUCUGAGCUGGACCCUCUCUCUACCUCUGGUGCAGAACUACAGCGGAAGGAAUCUCUG >hsa-mir-4316 MI0015845 (SEQ ID NO: 1880)AGUGGCCCAGGGUGAGGCUAGCUGGUGUGGUCACCCACUCUCCAGCCCAGCCCCAAUCCCACCACAACCAC >hsa-mir-4317 MI0015850 (SEQ ID NO: 1881)AAAAGGCGAGACAUUGCCAGGGAGUUUAUUUUGUAGCUCUCUUGAUAAAAUGUUUUAGCAAACAC >hsa-mir-4318 MI0015847 (SEQ ID NO: 1882)GCUUCUUAAUUAUGUCAUAAACCCACUGUGGACAAGGGCCUUGUCUUAGACAGUCACUGUGGGUACAUGCUAGGUGCUCAA >hsa-mir-4319 MI0015848 (SEQ ID NO: 1883)UUGGCUUGAGUCCCUGAGCAAAGCCACUGGGAAUGCUCCCUGAGGACGUUAUAUGAGUGCUCAGCUCAUGGGGCUAUGAUGGUCA >hsa-mir-4320 MI0015849 (SEQ ID NO: 1884)GACAUGUGGGGUUUGCUGUAGACAUUUCAGAUAACUCGGGAUUCUGUAGCUUCCUGGCAACUUUG >hsa-mir-4321 MI0015852 (SEQ ID NO: 1885)CUGGUCUCCGCAGAGCCUCUGCCCCUCCCGAGACACCCGCUACCUGGUGUUAGCGGUGGACCGCCCUGCGGGGGCCUGGC >hsa-mir-4322 MI0015851 (SEQ ID NO: 1886)ACCGCGAGUUCCGCGCCUGGCCGUGUCGCCCCACGAGGGGGACUGUGGGCUCAGCGCGUGGGGCCCGGAGCAU >hsa-mir-4323 MI0015853 (SEQ ID NO: 1887)CGGGGCCCAGGCGGGCAUGUGGGGUGUCUGGAGACGCCAGGCAGCCCCACAGCCUCAGACCUCGGGCAC >hsa-mir-4324 MI0015854 (SEQ ID NO: 1888)CGGCCCCUUUGUUAAGGGUCUCAGCUCCAGGGAACUUUAAAACCCUGAGACCCUAACCUUAAAGGUGCUGCA >hsa-mir-4325 MI0015865 (SEQ ID NO: 1889)GGGGAAGAUGUUGCACUUGUCUCAGUGAGAGAUGCUUCUAGAUCCAGGAGGCAGACCUCAAGGAUGGAGAGAAGGCAGAUCCUUUGAGAU >hsa-mir-4326 MI0015866 (SEQ ID NO: 1890)GCUGCUCUGCUGUUCCUCUGUCUCCCAGACUCUGGGUGGAUGGAGCAGGUCGGGGGCCA >hsa-mir-4327 MI0015867 (SEQ ID NO: 1891)GGCCUGGGUAGGCUUGCAUGGGGGACUGGGAAGAGACCAUGAACAGGUUAGUCCAGGGAGUUCUCAUCAAGCCUUUACUCAGUAG >hsa-mir-4328 MI0015904 (SEQ ID NO: 1892)AACAGUUGAGUCCUGAGAACCAUUGAGAACCAGUUUUCCCAGGAUUAACUGUUCCG >hsa-mir-4329 MI0015901 (SEQ ID NO: 1893)UAGAGAGGAAGGUGUACCAGGGUUUUGGAGUUUUUUUUUCCUCCUGAGACCCUAGUUCCACAUUCUGGAGC >hsa-mir-4330 MI0015902 (SEQ ID NO: 1894)AAUUGUCAGCAGGCAAUUAUCUGAGGAUGCAGGAGAGGAAGGGGGCUUCUUUUUGACGCCUACUUCAUCAGCUGCUCCUCAGAUCAGAGCCUUGCAGGUCAGGCC >hsa-mir-4417 MI0016753 (SEQ ID NO: 1895)GAAAACAACCAGGUGGGCUUCCCGGAGGGCGGAACACCCAGCCCCAGCAUCCAGGGCUCACCUACCACGUUUG >hsa-mir-4418 MI0016754 (SEQ ID NO: 1896)UGGUUUUUGCUCUGAGUGACCGUGGUGGUUGUGGGAGUCACUGCAGGACUCAGCAGGAAUUC >hsa-mir-4419a MI0016755 (SEQ ID NO: 1897)UGGUGGUGUGUGCCUGUAGUCUUAGCUACUCGGGAGGCUGAGGGAGGAGACUGCAGUGAGUGGAGGUCACGCCACUG >hsa-mir-4419b MI0016861 (SEQ ID NO: 1898)CUCAGGCUCAGUGGUGCAUGCUUAUAGUCCCAGCCACUCUGGAGGCUGAAGGAAGAUGGCUUGAGCCU >hsa-mir-4420 MI0016757 (SEQ ID NO: 1899)CUCUUGGUAUGAACAUCUGUGUGUUCAUGUCUCUCUGUGCACAGGGGACGAGAGUCACUGAUGUCUGUAGCUGAGAC >hsa-mir-4421 MI0016758 (SEQ ID NO: 1900)CUGGGUCUCCUUUCUGCUGAGAGUUGAACACUUGUUGGGACAACCUGUCUGUGGAAAGGAGCUACCUAC >hsa-mir-4422 MI0016759 (SEQ ID NO: 1901)AGUUCUUCUGCAGACAAAAGCAUCAGGAAGUACCCACCAUGUACCAGUGGGCCCUUCUUGAUGCUCUUGAUUGCAGAGGAGCC >hsa-mir-4423 MI0016760 (SEQ ID NO: 1902)AUCAUGUACUGCAGUUGCCUUUUUGUUCCCAUGCUGUUUAAGCCUAGCAUAGGCACCAAAAAGCAACAACAGUAUGUGAA >hsa-mir-4424 MI0016763 (SEQ ID NO: 1903)CUUACAUCACACACAGAGUUAACUCAAAAUGGACUAAUUUUUCCACUAGUUAGUCCAUUUCAAGUUAACUCUGUGUGUGAUGUAGU >hsa-mir-4425 MI0016764 (SEQ ID NO: 1904)GUGCUUUACAUGAAUGGUCCCAUUGAAUCCCAACAGCUUUGCGAAGUGUUGUUGGGAUUCAGCAGGACCAUUCGUGUAAAGUAA >hsa-mir-4426 MI0016765 (SEQ ID NO: 1905)AGUUGGAAGAUGGACGUACUUUGUCUGACUACAAUAUUCAAAAGGAGUCUACUCUUCAUCUUG >hsa-mir-4427 MI0016766 (SEQ ID NO: 1906)GAAGCCUCUUGGGGCUUAUUUAGACAAUGGUUUCAUCAUUUCGUCUGAAUAGAGUCUGAAGAGUCUUU >hsa-mir-4428 MI0016767 (SEQ ID NO: 1907)UUGGCAGGUGCCAUGUUGCCUGCUCCUUACUGUACACGUGGCUGGCAAGGAGACGGGAACAUGGAGCCGCCAU >hsa-mir-4429 MI0016768 (SEQ ID NO: 1908)AGGGAGAAAAGCUGGGCUGAGAGGCGACUGGUGUCUAAUUUGUUUGUCUCUCCAACUCAGACUGCCUGGCCCA >hsa-mir-4430 MI0016769 (SEQ ID NO: 1909)GUGAGGCUGGAGUGAGCGGAGAUCGUACCACUGCACUCCAACCUGGUGA >hsa-mir-4431 MI0016771 (SEQ ID NO: 1910)UGGUUUGCGACUCUGAAAACUAGAAGGUUUAUGACUGGGCAUUUCUCACCCAAUGCCCAAUAUUGAACUUUCUAGUUGUCAGAGUCAUUAACCC >hsa-mir-4432 MI0016772 (SEQ ID NO: 1911)GCAUCUUGCAGAGCCGUUCCAAUGCGACACCUCUAGAGUGUCAUCCCCUAGAAUGUCACCUUGGAAAGACUCUGCAAGAUGCCU >hsa-mir-4433 MI0016773 (SEQ ID NO: 1912)CAUCCUCCUUACGUCCCACCCCCCACUCCUGUUUCUGGUGAAAUAUUCAAACAGGAGUGGGGGUGGGACAUAAGGAGGAUA >hsa-mir-4434 MI0016774 (SEQ ID NO: 1913)UCACUUUAGGAGAAGUAAAGUAGAACUUUGGUUUUCAACUUUUCCUACAGUGU >hsa-mir-4435-1 MI0016775 (SEQ ID NO: 1914)AGGCAGCAAAUGGCCAGAGCUCACACAGAGGGAUGAGUGCACUUCACCUGCAGUGUGACUCAGCAGGCCAACAGAUGCUA >hsa-mir-4435-2 MI0016777 (SEQ ID NO: 1915)GCAAAUGGCCAGAGCUCACACAGAGGGAUGAGUGCACUUCACCUGCAGUGUGACUCAGCAGGCCAACAGAUGCU >hsa-mir-4436a MI0016776 (SEQ ID NO: 1916)GCCUCACUUUUCCACUUAUGCCUGCCCUGCCCCUCGAAUCUGCUCCACGAUUUGGGCAGGACAGGCAGAAGUGGAUAAGUGAGGA >hsa-mir-4436b-1 MI0017425 (SEQ ID NO: 1917)GUGUCCUCACUUGUCCACUUCUGCCUGCCCUGCCCAAAUGGUGGAGCAGAUUCGAGGGGCAGGGCAGGAAGAAGUGGACAAGUGAGGCCAU >hsa-mir-4436b-2 MI0019110 (SEQ ID NO: 1918)GUGUCCUCACUUGUCCACUUCUGCCUGCCCUGCCCAAAUGGUGGAGCAGAUUCGAGGGGCAGGGCAGGAAGAAGUGGACAAGUGAGGCCAU >hsa-mir-4437 MI0016778 (SEQ ID NO: 1919)ACUUUGUGCAUUGGGUCCACAAGGAGGGGAUGACCCUUGUGGGCUCAGGGUACAAAGGUU >hsa-mir-4438 MI0016781 (SEQ ID NO: 1920)UAAGUGUAAACUUAAGGACUGUCUUUUCUAAGCCUGUGCCUUGCCUUUCCUUUGGCACAGGCUUAGAAAAGACAGUCUUUAAGUUUACACUUC >hsa-mir-4439 MI0016782 (SEQ ID NO: 1921)CCAGUGACUGAUACCUUGGAGGCAUUUUAUCUAAGAUACACACAAAGCAAAUGCCUCUAAGGUAUCAGUUUACCAGGCCA >hsa-mir-4440 MI0016783 (SEQ ID NO: 1922)CUCUCACCAAGCAAGUGCAGUGGGGCUUGCUGGCUUGCACCGUGACUCCCUCUCACCAAGCAAGUGUCGUGGGGCUUGCUGGCUUGCACUGUGAAGAU >hsa-mir-4441 MI0016784 (SEQ ID NO: 1923)CAGAGUCUCCUUCGUGUACAGGGAGGAGACUGUACGUGAGAGAUAGUCAGAUCCGCAUGUUAGAGCAGAGUCUCCUUCGUGUACAGGGAGGAGAUUGUAC >hsa-mir-4442 MI0016785 (SEQ ID NO: 1924)GCGCCCUCCCUCUCUCCCCGGUGUGCAAAUGUGUGUGUGCGGUGUUAUGCCGGACAAGAGGGAGGUG >hsa-mir-4443 MI0016786 (SEQ ID NO: 1925)GGUGGGGGUUGGAGGCGUGGGUUUUAGAACCUAUCCCUUUCUAGCCCUGAGCA >hsa-mir-4444-1 MI0016787 (SEQ ID NO: 1926)GUGACGACUGGCCCCGCCUCUUCCUCUCGGUCCCAUAUUGAACUCGAGUUGGAAGAGGCGAGUCCGGUCUCAAA >hsa-mir-4444-2 MI0019111 (SEQ ID NO: 1927)GUGACGACUGGCCCCGCCUCUUCCUCUCGGUCCCAUAUUGAACUCGAGUUGGAAGAGGCGAGUCCGGUCUCAAA >hsa-mir-4445 MI0016788 (SEQ ID NO: 1928)UUCCUGCAGAUUGUUUCUUUUGCCGUGCAAGUUUAAGUUUUUGCACGGCAAAAGAAACAAUCCAGAGGGU >hsa-mir-4446 MI0016789 (SEQ ID NO: 1929)CUGGUCCAUUUCCCUGCCAUUCCCUUGGCUUCAAUUUACUCCCAGGGCUGGCAGUGACAUGGGUCAA >hsa-mir-4447 MI0016790 (SEQ ID NO: 1930)GUUCUAGAGCAUGGUUUCUCAUCAUUUGCACUACUGAUACUUGGGGUCAGAUAAUUGUUUGUGGUGGGGGCUGUUGUUUGCAUUGUAGGAU >hsa-mir-4448 MI0016791 (SEQ ID NO: 1931)AGGAGUGACCAAAAGACAAGAGUGCGAGCCUUCUAUUAUGCCCAGACAGGGCCACCAGAGGGCUCCUUGGUCUAGGGGUAAUGCCA >hsa-mir-4449 MI0016792 (SEQ ID NO: 1932)AGCAGCCCUCGGCGGCCCGGGGGGCGGGCGGCGGUGCCCGUCCCGGGGCUGCGCGAGGCACAGGCG >hsa-mir-4450 MI0016795 (SEQ ID NO: 1933)UGUCUGGGGAUUUGGAGAAGUGGUGAGCGCAGGUCUUUGGCACCAUCUCCCCUGGUCCCUUGGCU >hsa-mir-4451 MI0016797 (SEQ ID NO: 1934)UCUGUACCUCAGCUUUGCUCCCAACCAACCACUUCCACAUGUUUUGCUGGUAGAGCUGAGGACAGC >hsa-mir-4452 MI0016798 (SEQ ID NO: 1935)UGGAUCACUUGAGGCCAAGAGUGCAAGGCUGUAGUGUGCACAGCCUUGAAUUCUUGGCCUUAAGUGAUCCC >hsa-mir-4453 MI0016799 (SEQ ID NO: 1936)UGGAGAGCUUGGUCUGUAGCGGUUUCCUUCGGGGCAGGUGGGGACUGCUCCUUUGGGAGGAAGGAGGAGGCCCAGGCCGCGUCUUCAGG >hsa-mir-4454 MI0016800 (SEQ ID NO: 1937)CCGGAUCCGAGUCACGGCACCAAAUUUCAUGCGUGUCCGUGUGAAGAGACCACCA >hsa-mir-4455 MI0016801 (SEQ ID NO: 1938)AGAAGGGUGUGUGUGUUUUUCCUGAGAAUAAGAGAAGGAAGGACAGCCAAAUUCUUCA >hsa-mir-4456 MI0016802 (SEQ ID NO: 1939)AUGAACCUGGUGGCUUCCUUUUCUGGGAGGAAGUUAGGGUUCA >hsa-mir-4457 MI0016803 (SEQ ID NO: 1940)GGAGUACUCCAGUCAAUACCGUGUGAGUUAGAAAAGCUCAAUUCACAAGGUAUUGACUGGCGUAUUCA >hsa-mir-4458 MI0016804 (SEQ ID NO: 1941)GAGCGCACAGAGGUAGGUGUGGAAGAAAGUGAAACACUAUUUUAGGUUUUAGUUACACUCUGCUGUGGUGUGCUG >hsa-mir-4459 MI0016805 (SEQ ID NO: 1942)ACCCAGGAGGCGGAGGAGGUGGAGGUUGCAGUGAGCCAAGAUCGUGGCACUGACUCCAGCCUGGGG >hsa-mir-4460 MI0016806 (SEQ ID NO: 1943)GUUUUUUGCCCAUAGUGGUUGUGAAUUUACCUUCUCCUCUUUGCAGUGAUAAAGGAGGUAAAUUCACAACCACUGUGGGCAGAAAC >hsa-mir-4461 MI0016807 (SEQ ID NO: 1944)GAGUAGGCUUAGGUUAUGUACGUAGUCUAGGCCAUACGUGUUGGAGAUUGAGACUAGUAGGGCUAGGCCUACUG >hsa-mir-4462 MI0016810 (SEQ ID NO: 1945)CUUCCCAGCUGCCCUAAGUCAGGAGUGGCUUUCCUGACACGGAGGGUGGCUUGGGAAA >hsa-mir-4463 MI0016811 (SEQ ID NO: 1946)AAUAGAUUAUUGGUCACCACCUCCAGUUUCUGAAUUUGUGAGACUGGGGUGGGGCCUGAGAAUUUGC >hsa-mir-4464 MI0016812 (SEQ ID NO: 1947)GGAACCUUAGUAAGGUUUGGAUAGAUGCAAUAAAGUAUGUCCACAGCUGAAAGGACAUACUUUAUUGCAUGUAUCCAAACCUUACUAAUUCA >hsa-mir-4465 MI0016816 (SEQ ID NO: 1948)CAUGUGUCCCCUGGCACGCUAUUUGAGGUUUACUAUGGAACCUCAAGUAGUCUGACCAGGGGACACAUGA >hsa-mir-4466 MI0016817 (SEQ ID NO: 1949)ACGCGGGUGCGGGCCGGCGGGGUAGAAGCCACCCGGCCCGGCCCGGCCCGGCGA >hsa-mir-4467 MI0016818 (SEQ ID NO: 1950)UGGUGGCGGCGGUAGUUAUGGGCUUCUCUUUCUCACCAGCAGCCCCUGGGCCGCCGCCUCCCU >hsa-mir-4468 MI0016819 (SEQ ID NO: 1951)AGUCUUCUCCUGGGGCUUUGGUGGCUAUGGUUGACUGGGCCACUCAGAGCAGAAGGAUGAGAUG >hsa-mir-4469 MI0016820 (SEQ ID NO: 1952)CCGACGCGGAGAGCGGCUCUAGGUGGGUUUGGCGGCGGCGAGGACACCGCCGCUCCCUCUAGGGUCGCUCGGAGCGUGA >hsa-mir-4470 MI0016821 (SEQ ID NO: 1953)CGAGCCUCUUUCGGCUUUCCAGUUUGUCUCGGUCCUUUGGAACGUGGCAAACGUGGAAGCCGAGAGGGCUCU >hsa-mir-4471 MI0016822 (SEQ ID NO: 1954)CCAAAUUUAAAACUUAAACCUCUACUAAGUUUCCAUGAAAAGAACCCAUGGGAACUUAGUAGAGGUUUAAGUUUUAAAUUUGA >hsa-mir-4472-1 MI0016823 (SEQ ID NO: 1955)UGGCAGACCCUUGCUCUCUCACUCUCCCUAAUGGGGCUGAAGACAGCUCAGGGGCAGGGUGGGGGGUGUUGUUUUUGUUU >hsa-mir-4472-2 MI0016824 (SEQ ID NO: 1956)UGGUGGGGGUGGGGGGUGUUGUUUUUGUUUUUGAGACAGAGUCUUGCUCCGUCGCCCAGGCCGGAGU >hsa-mir-4473 MI0016825 (SEQ ID NO: 1957)AAGGAACAGGGGACACUUGUAAUGGAGAACACUAAGCUAUGGACUGCUAUGGACUGCUAGUGCUCUCCGUUACAAGUAUCCCCUGUUACCU >hsa-mir-4474 MI0016826 (SEQ ID NO: 1958)UUGCCUACCUUGUUAGUCUCAUGAUCAGACACAAAUAUGGCUCUUUGUGGCUGGUCAUGAGGCUAACAAGGUAGGCAC >hsa-mir-4475 MI0016827 (SEQ ID NO: 1959)AUCUCAAUGAGUGUGUGGUUCUAAAUGACUCAUAGUCAAGGGACCAAGCAUUCAUUAUGAA >hsa-mir-4476 MI0016828 (SEQ ID NO: 1960)AAAAGCCUGUCCCUAAGUCCCUCCCAGCCUUCCAGAGUUGGUGCCAGGAAGGAUUUAGGGACAGGCUUUG >hsa-mir-4477a MI0016829 (SEQ ID NO: 1961)UCCUCCUCCCAUCAAUCACAAAUGUCCUUAAUGGCAUUUAAGGAUUGCUAUUAAGGACAUUUGUGAUUCACGGGAGGAGGU >hsa-mir-4477b MI0016830 (SEQ ID NO: 1962)ACCUCCUCCCGUGAAUCACAAAUGUCCUUAAUAGCAAUCCUUAAAUGCCAUUAAGGACAUUUGUGAUUGAUGGGAGGAGGA >hsa-mir-4478 MI0016831 (SEQ ID NO: 1963)GGCCGAGGCUGAGCUGAGGAGCCUCCAAACCUGUAGACAGGGUCAUGCAGUACUAGGGGCGAGCCUCAUCCCCUGCAGCCCUGGCC >hsa-mir-4479 MI0016838 (SEQ ID NO: 1964)GAAACCAAGUCCGAGCGUGGCUGGCGCGGGAAAGUUCGGGAACGCGCGCGGCCGUGCUCGGAGCAGCGCCA >hsa-mir-4480 MI0016841 (SEQ ID NO: 1965)GCAGAGGUGAGUUGACCUCCACAGGGCCACCCAGGGAGUAAGUAGCCAAGUGGAAGUUACUUUACCUCUGU >hsa-mir-4481 MI0016842 (SEQ ID NO: 1966)GGAGUGGGCUGGUGGUUUUUUAAGAGGAAGGGAGACCUAAGCUAGCACAUGAGCACGCUC >hsa-mir-4482-1 MI0016843 (SEQ ID NO: 1967)AGUGAGCAACCCAGUGGGCUAUGGAAAUGUGUGGAAGAUGGCAUUUCUAUUUCUCAGUGGGGCUCUUACC >hsa-mir-4482-2 MI0017872 (SEQ ID NO: 1968)AAUAAAAAUUGAAUAGUGAGCAACCCAGUGGGCUAUGGAAAUGUGUGGAAGAUGGCAUUUCUAUUUCUCAGUGGGGCUCUUACCUAUUACUCAUCAAC >hsa-mir-4483 MI0016844 (SEQ ID NO: 1969)AAAAAACAACAUACUUAGUGCAUACCCAUAUAAUAUUAGGGGUGGUCUGUUGUUGUUUUUCU >hsa-mir-4484 MI0016845 (SEQ ID NO: 1970)GGGUUUCCUCUGCCUUUUUUUCCAAUGAAAAUAACGAAACCUGUUAUUUCCCAUUGAGGGGGAAAAAGGCGGGAGAAGCCCCA >hsa-mir-4485 MI0016846 (SEQ ID NO: 1971)AGAGGCACCGCCUGCCCAGUGACAUGCGUUUAACGGCCGCGGUACCCUAACUGUGCA >hsa-mir-4486 MI0016847 (SEQ ID NO: 1972)GCAUGCUGGGCGAGGCUGGCAUCUAGCACAGGCGGUAGAUGCUUGCUCUUGCCAUUGCAAUGA >hsa-mir-4487 MI0016848 (SEQ ID NO: 1973)ACUGUCCUUCAGCCAGAGCUGGCUGAAGGGCAGAAGGGAACUGUCCUUCAGCCAGAGCUGGCUGAAGGGCAGA >hsa-mir-4488 MI0016849 (SEQ ID NO: 1974)GGUAGGGGGCGGGCUCCGGCGCUGGGACCCCACUAGGGUGGCGCCUUGGCCCCGCCCCGCCC >hsa-mir-4489 MI0016850 (SEQ ID NO: 1975)GGGGGUGGGGCUAGUGAUGCAGGACGCUGGGGACUGGAGAAGUCCUGCCUGACCCUGUCCCA >hsa-mir-4490 MI0016852 (SEQ ID NO: 1976)AUAGUUUCUGCAAUGCUCAAAUCUCUGGCCAAAGACCAGAACUUAAUGGUCUCUGGUAAGAGAUUUGGGCAUAUUAGAAACUAA >hsa-mir-4491 MI0016853 (SEQ ID NO: 1977)ACAUUUGGUCACACCAGUCCACAUUAACGUGGACCAGACAAUAUUAAUGUGGACUGGUGUGACCAAAA >hsa-mir-4492 MI0016854 (SEQ ID NO: 1978)CUGCAGCGUGCUUCUCCAGGCCCCGCGCGCGGACAGACACACGGACAAGUCCCGCCAGGGGCUGGGCGCGCGCCAGCCGG >hsa-mir-4493 MI0016855 (SEQ ID NO: 1979)CCAGAGAUGGGAAGGCCUUCCGGUGAUUAUCACAGCCAUGCCUUUACCUCCAGAAGGCCUUUCCAUCUCUGUC >hsa-mir-4494 MI0016856 (SEQ ID NO: 1980)AGUUUUAGUUACCCUGGUCAUCUGCAGUCUGAAAAUACAAAAUGGAAAAUUCCAGACUGUGGCUGACCAGAGGUAACUGAAACC >hsa-mir-4495 MI0016857 (SEQ ID NO: 1981)AAGAAAUGUAAACAGGCUUUUUGCUCAGUGGAGUUAUUUUGAGCAAAAAGCUUAUUUACAUUUCUG >hsa-mir-4496 MI0016858 (SEQ ID NO: 1982)ACAUCAGCUCAUAUAAUCCUCGAAGCUGCCUUUAGAAAUGAGGAAACUGAAGCUGAGAGGG >hsa-mir-4497 MI0016859 (SEQ ID NO: 1983)ACCUCCGGGACGGCUGGGCGCCGGCGGCCGGGAGAUCCGCGCUUCCUGAAUCCCGGCCGGCCCGCCCGGCGCCCGUCCGCCCGCGGGUC >hsa-mir-4498 MI0016860 (SEQ ID NO: 1984)AGGGCUGGGCUGGCAGGGCAAGUGCUGCAGAUCUUUGUCUAAGCAGCCCCUGCCUUGGAUCUCCCA >hsa-mir-4499 MI0016862 (SEQ ID NO: 1985)AAGACUGAGAGGAGGGAACUGGUGAGUUGUACAUAGAAAUGCUUUCUAACUCCUUGUCUCAGUCUGUUU >hsa-mir-4500 MI0016863 (SEQ ID NO: 1986)CAGGAGAGAAAGUACUGCCCAGAAGCUAAAGUGUAGAUCAAACGCAUAAUGGCUGAGGUAGUAGUUUCUUGAACUU >hsa-mir-4501 MI0016864 (SEQ ID NO: 1987)UAUGUGACCUCGGAUGAAUCACUGAAAUAUGUCUGAGCUUCUGUUUCAUCAGAUGUCACAUUUU >hsa-mir-4502 MI0016865 (SEQ ID NO: 1988)AGCCUUUAGCAAGUUGUAAUCUUUUUGCUGAUGGAGGGUCUUGCCUCCAUGGGGAUGGCUGAUGAUGAUGGUGCUGAAGGC >hsa-mir-4503 MI0016866 (SEQ ID NO: 1989)ACAAUGUAGAUAUUUAAGCAGGAAAUAGAAUUUACAUAUAAAUUUCUAUUUGUUUCUAUUUCCUGCUUAAAUAUCUACAUUGC >hsa-mir-4504 MI0016867 (SEQ ID NO: 1990)CUAAGAUAAUGUCCUCCAGGUUCAUCUCUGUUGUCAUUUGUGGCAUGGACCAUUUGUGACAAUAGAGAUGAACAUGGAGGAUAUUAUCUUAA >hsa-mir-4505 MI0016868 (SEQ ID NO: 1991)GGAGGCUGGGCUGGGACGGACACCCGGCCUCCACUUUCUGUGGCAGGUACCUCCUCCAUGUCGGCCCGCCUUG >hsa-mir-4506 MI0016869 (SEQ ID NO: 1992)UGGCCUCUGCCAUCAGACCAUCUGGGUUCAAGUUUGGCUCCAUCUUUAUGAAAUGGGUGGUCUGAGGCAAGUGGUCU >hsa-mir-4507 MI0016871 (SEQ ID NO: 1993)UCUGGGCUGAGCCGAGCUGGGUUAAGCCGAGCUGGGUUGGGCUGGGCUGGGU >hsa-mir-4508 MI0016872 (SEQ ID NO: 1994)AGGACCCAGCGGGGCUGGGCGCGCGGAGCAGCGCUGGGUGCAGCGCCUGCGCCGGCAGCUGCAAGGGCCG >hsa-mir-4509-1 MI0016873 (SEQ ID NO: 1995)CUUUAAUACUAUCUCAAACUAAAGGAUAUAGAAGGUUUUCCCUUUCUCUUGCCCUGAAACCUUCUGUAUCCUUUAUUUUGAGAUAGUAUUAGAA >hsa-mir-4509-2 MI0016874 (SEQ ID NO: 1996)CUUUAAUACUAUCUCAAACUAAAGGAUAUAGAAGGUUUUCCCUUUCUCUUGCCCUGAAACCUUCUGUAUCCUUUAUUUUGAGAUAGUAUUAGAA >hsa-mir-4509-3 MI0016875 (SEQ ID NO: 1997)CUUUAAUACUAUCUCAAACUAAAGGAUAUAGAAGGUUUUCCCUUUCUCUUGCCCUGAAACCUUCUGUAUCCUUUAUUUUGAGAUAGUAUUAGAA >hsa-mir-4510 MI0016876 (SEQ ID NO: 1998)GUGUAUGUGAGGGAGUAGGAUGUAUGGUUGUUAGAUAGACAACUACAAUCUUUUCUCACAACAGACAG >hsa-mir-4511 MI0016877 (SEQ ID NO: 1999)AAAAAAAAGGGAAAGAAGAACUGUUGCAUUUGCCCUGCACUCAGUUUGCACAGGGUAAAUGCAAUAGUUCUUCUUUCCCUUUUUUUA >hsa-mir-4512 MI0016878 (SEQ ID NO: 2000)CUCAGCCCGGGCAAUAUAGUGAGACCUCGUCUCUACAAAAAAUUGAGACAGGGCCUCACUGUAUCGCCCAGGCUGGA >hsa-mir-4513 MI0016879 (SEQ ID NO: 2001)AUUCUAGGUGGGGAGACUGACGGCUGGAGGCCCAUAAGCUGUCUAAAACUUCGGCCCCCAGAUUUCUGGUCUCCCCACUUCAGAAC >hsa-mir-4514 MI0016880 (SEQ ID NO: 2002)GUUGAGACAGGCAGGAUUGGGGAAACAUCUUUUACCUCGUCUCUUGCCUGUUUUAGA >hsa-mir-4515 MI0016881 (SEQ ID NO: 2003)GCGGGAGGUGUAACAGGACUGGACUCCCGGCAGCCCCAGGGCAGGGGCGUGGGGAGCUGGUCCUAGCUCAGCGCUCCCGGA >hsa-mir-4516 MI0016882 (SEQ ID NO: 2004)AGGGAGAAGGGUCGGGGCAGGGAGGGCAGGGCAGGCUCUGGGGUGGGGGGUCUGUGAGUCAGCCACGGCUCUGCCCACGUCUCCCC >hsa-mir-4517 MI0016883 (SEQ ID NO: 2005)AGGUAAAUAUGAUGAAACUCACAGCUGAGGAGCUUAGCAAGUAGCUAAGGCCAGAGCUUGUGUUUGGGUGGUGUGGCUG >hsa-mir-4518 MI0016884 (SEQ ID NO: 2006)UGGGGGAAAAGUGCUGGGAUUGAUUAGUGAUGUCUGCUGGGGAACCGGGGCUCAGGGAUGAUAACUGUGCUGAGAAGCCCCCU >hsa-mir-4519 MI0016885 (SEQ ID NO: 2007)AACCUCAGCAGUGCGCAGGGCUGCACUGUCUCCGUCUGCGGCCUGCAGUAAGCGGGUA >hsa-mir-4520a MI0016886 (SEQ ID NO: 2008)GUGUGCCACCUGCGUGUUUUCUGUCCAAAUCAGAAAAGGAUUUGGACAGAAAACACGCAGGAAGAAGGAA >hsa-mir-4520b MI0017358 (SEQ ID NO: 2009)CCUGCGUGUUUUCUGUCCAAAUCCUUUUCUGAUUUGGACAGAAAACACGCAGGU >hsa-mir-4521 MI0016887 (SEQ ID NO: 2010)UCGGCUAAGGAAGUCCUGUGCUCAGUUUUGUAGCAUCAAAACUAGGAUUUCUCUUGUUAC >hsa-mir-4522 MI0016889 (SEQ ID NO: 2011)GCGGGCGUUGCCUGGGGGCCUCGCAGGGGGAGAUCCAGCCCAGGCUGGUUCCGCUGACUCUGCCUGUAGGCCGGUGGCGUCUUCUGG >hsa-mir-4523 MI0016890 (SEQ ID NO: 2012)GCGGGGGACCGAGAGGGCCUCGGCUGUGUGAGGACUAGAGGCGGCCGAGGCCCGGGCCGGUUCCCCCGA >hsa-mir-4524a MI0016891 (SEQ ID NO: 2013)GAACGAUAGCAGCAUGAACCUGUCUCACUGCAGAAUUAUUUUGAGACAGGCUUAUGCUGCUAUCCUUCA >hsa-mir-4524b MI0019114 (SEQ ID NO: 2014)UAGCUGGGUGGAUGUGUUCUUUUGAAGGAUAGCAGCAUAAGCCUGUCUCAAAAUAAUUCUGCAGUGAGACAGGUUCAUGCUGCUAUCGUUCCAAAGAGGAAGGGUAAUCACUGUC >hsa-mir-4525 MI0016892 (SEQ ID NO: 2015)GUCAGAGGGGGGAUGUGCAUGCUGGUUGGGGUGGGCUGCCUGUGGACCAAUCAGCGUGCACUUCCCCACCCUGAA >hsa-mir-4526 MI0016893 (SEQ ID NO: 2016)UGCGGUGACAUCAGGGCCCAGUCCCUGCUGUCAUGCCCCAGGUGACGUGCUGGGCUGACAGCAGGGCUGGCCGCUAACGUCACUGUC >hsa-mir-4527 MI0016894 (SEQ ID NO: 2017)CCAGAAGUGGUCUGCAAAGAGAUGACUGUGAAUCCAAGAUCCACAUCAGCUCUGUGCUGCCUACAUCUGA >hsa-mir-4528 MI0016895 (SEQ ID NO: 2018)UAUUCUACUGAGAGUACAGAUCUUUAUAUAUAUGAUCAUUAUAUGUAUGAUGAGAUCAUUAUAUGUAUGAUCUGGACACCCAGUAGAAUC >hsa-mir-4529 MI0016896 (SEQ ID NO: 2019)AUGACAGGCCAUCAGCAGUCCAAUGAAGACAUGAAGACCCAAUGUCUUCAUUGGACUGCUGAUGGCCCGUCACUGGGA >hsa-mir-4530 MI0016897 (SEQ ID NO: 2020)CGACCGCACCCGCCCGAAGCUGGGUCAAGGAGCCCAGCAGGACGGGAGCGCGGCGC >hsa-mir-4531 MI0016898 (SEQ ID NO: 2021)GCCUAGGAGUCCUUGGUCAGUGGGGACAUGGAGAAGGCUUCUGAGGA >hsa-mir-4532 MI0016899 (SEQ ID NO: 2022)ACAGACCCCGGGGAGCCCGGCGGUGAAGCUCCUGGUAUCCUGGGUGUCUGA >hsa-mir-4533 MI0016900 (SEQ ID NO: 2023)UGAGAAUGUGGAAGGAGGUUGCCGGACGCUGCUGGCUGCCUUCCAGCGUCCACUUCCCUUUCUCUCUCUCC >hsa-mir-4534 MI0016901 (SEQ ID NO: 2024)UGUGAAUGACCCCCUUCCAGAGCCAAAAUCACCAGGGAUGGAGGAGGGGUCUUGGGUACU >hsa-mir-4535 MI0016903 (SEQ ID NO: 2025)AACUGGGUCCCAGUCUUCACAGUUGGUUUCUGACACGUGGACCUGGCUGGGACGAUGUG >hsa-mir-4536-1 MI0016906 (SEQ ID NO: 2026)AUGUGGUAGAUAUAUGCACGAUAUAUAUACUGCCCUGCUUUUAUACAUACAUACAUACAUACCUAUAUCGUGCAUAUAUCUACCACAU >hsa-mir-4536-2 MI0019149 (SEQ ID NO: 2027)AUGUGGUAGAUAUAUGCACGAUAUAGGUAUGUAUGUAUGUAUGUAUAAAAGCAGGGCAGUAUAUAUAUCGUGCAUAUAUCUACCACAU >hsa-mir-4537 MI0016908 (SEQ ID NO: 2028)UGAGCCGAGCUGAGCUUAGCUGGGCUGAGCUAACCAGGGCUGGGCUGAGCUGGGCUGAGCUGAGCUGAGC >hsa-mir-4538 MI0016909 (SEQ ID NO: 2029)GAGCUUGGAUGAGCUGGGCUGAACUGGGCUGGGUUGAGCUGGGCUGGGCUGAGUUGAGCCAGGCUGAUCUGGGCUGAG >hsa-mir-4539 MI0016910 (SEQ ID NO: 2030)UGAGCUGGGCUCUGCUGUGCUGUGCUGAGCAGGGCUGAGCUGAACUGGGCUGAGCUGGGC >hsa-mir-4540 MI0016911 (SEQ ID NO: 2031)AAGCUGCAUGGACCAGGACUUGGCACCUUUGGCCUUAGUCCUGCCUGUAGGUUUA >hsa-mir-4632 MI0017259 (SEQ ID NO: 2032)GAGGGCAGCGUGGGUGUGGCGGAGGCAGGCGUGACCGUUUGCCGCCCUCUCGCUGCUCUAG >hsa-mir-4633 MI0017260 (SEQ ID NO: 2033)UGGCAAGUCUCCGCAUAUGCCUGGCUAGCUCCUCCACAAAUGCGUGUGGAGGAGCUAGCCAGGCAUAUGCAGAGCGUCA >hsa-mir-4634 MI0017261 (SEQ ID NO: 2034)GGACAAGGGCGGCGCGACCGGCCCGGGGCUCUUGGGCGGCCGCGUUUCCCCUCC >hsa-mir-4635 MI0017262 (SEQ ID NO: 2035)CCGGGACUUUGUGGGUUCUGACCCCACUUGGAUCACGCCGACAACACUGGUCUUGAAGUCAGAACCCGCAAAGUCCUGG >hsa-mir-4636 MI0017263 (SEQ ID NO: 2036)UAGAUUCAGAACUCGUGUUCAAAGCCUUUAGCCCAGCAAUGGGAGAGUGCUAAAGGCUUCAAGCACGAGUUCUGAAUCUA >hsa-mir-4637 MI0017264 (SEQ ID NO: 2037)CCCUUACUUGGAUCUGCAAUUAGUAUUUUAAUCAUAGAUUGUAUUUAGUUAGUUUUUAAUACUAACUGCAGAUUCAAGUGAGGG >hsa-mir-4638 MI0017265 (SEQ ID NO: 2038)GACUCGGCUGCGGUGGACAAGUCCGGCUCCAGAACCUGGACACCGCUCAGCCGGCCGCGGCAGGGGUC >hsa-mir-4639 MI0017266 (SEQ ID NO: 2039)UUGCUAAGUAGGCUGAGAUUGAUGUCAGGUUAUCCCCAAGCAUAACCUCACUCUCACCUUGCUUUGCAG >hsa-mir-4640 MI0017267 (SEQ ID NO: 2040)CUGUGGGCUGGGCCAGGGAGCAGCUGGUGGGUGGGAAGUAAGAUCUGACCUGGACUCCAUCCCACCCACCCCCUGUUUCCUGGCCCACAG >hsa-mir-4641 MI0017268 (SEQ ID NO: 2041)GGGGGGCAGGGGGCAGAGGGCAUCAGAGGACAGCCGCCUGGUGCCCAUGCCAUACUUUUGCCUCAG >hsa-mir-4642 MI0017269 (SEQ ID NO: 2042)CACAACUGCAUGGCAUCGUCCCCUGGUGGCUGUGGCCUAGGGCAAGCCACAAAGCCACUCAGUGAUGAUGCCAGCAGUUGUG >hsa-mir-4643 MI0017270 (SEQ ID NO: 2043)GUGUGCCCUAGCAUUUAUAAUCAUGUGUUCAUUCACAUGAUCAUAAGUGGACACAUGACCAUAAAUGCUAAAGCACAC >hsa-mir-4644 MI0017271 (SEQ ID NO: 2044)GCGGCGGUGCUCUGCCUCUUUCUCCAUCCACCCUGGUCCAGGUCCACAGCAGUGGAGAGAGAAAAGAGACAGAAGGAUGGCCGU >hsa-mir-4645 MI0017272 (SEQ ID NO: 2045)UGAUAGGGAAACCAGGCAAGAAAUAUUGUCUCCUCAAGUUGCGACGAGACAGUAGUUCUUGCCUGGUUUCUCUAUCA >hsa-mir-4646 MI0017273 (SEQ ID NO: 2046)ACUGGGAAGAGGAGCUGAGGGACAUUGCGGAGAGGGUCUCACAUUGUCCCUCUCCCUUCCCAG >hsa-mir-4647 MI0017274 (SEQ ID NO: 2047)CCAGGAGGGUGAAGAUGGUGCUGUGCUGAGGAAAGGGGAUGCAGAGCCCUGCCCAGCACCACCACCUCCUAUGCUCCUGG >hsa-mir-4648 MI0017275 (SEQ ID NO: 2048)UGUGGGACUGCAAAUGGGAGCUCAGCACCUGCCUGCCACCCACGCAGACCAGCCCCUGCUCUGUUCCCACAG >hsa-mir-4649 MI0017276 (SEQ ID NO: 2049)UCUGGGCGAGGGGUGGGCUCUCAGAGGGGCUGGCAGUACUGCUCUGAGGCCUGCCUCUCCCCAG >hsa-mir-4650-1 MI0017277 (SEQ ID NO: 2050)UUCUGUAGAGAUUAUCAGGCCUCUUUCUACCUUCCAAGGCUCAGAAGGUAGAAUGAGGCCUGACAUAUCUGCAGGA >hsa-mir-4650-2 MI0017278 (SEQ ID NO: 2051)UUCUGUAGAGAUUAUCAGGCCUCUUUCUACCUUCCAAGGCUCAGAAGGUAGAAUGAGGCCUGACAUAUCUGCAGGA >hsa-mir-4651 MI0017279 (SEQ ID NO: 2052)CGGCGACGGCGGGGUGGGUGAGGUCGGGCCCCAAGACUCGGGGUUUGCCGGGCGCCUCAGUUCACCGCGGCCG >hsa-mir-4652 MI0017280 (SEQ ID NO: 2053)UAUUGGACGAGGGGACUGGUUAAUAGAACUAACUAACCAGAACUAUUUUGUUCUGUUAACCCAUCCCCUCAUCUAAUA >hsa-mir-4653 MI0017281 (SEQ ID NO: 2054)UUGUCCAAUUCUCUGAGCAAGGCUUAACACCAAAGGGUUAAGGGUUUGCUCUGGAGUUAAGGGUUGCUUGGAGAAUUGGAGAA >hsa-mir-4654 MI0017282 (SEQ ID NO: 2055)CUGGCUGGUUGUGGGAUCUGGAGGCAUCUGGGGUUGGAAUGUGACCCCAGUCUCCUUUUCCCUCAUCAUCUGCCAG >hsa-mir-4655 MI0017283 (SEQ ID NO: 2056)CCAAGGGCACACCGGGGAUGGCAGAGGGUCGUGGGAAAGUGUUGACCCUCGUCAGGUCCCCGGGGAGCCCCUGG >hsa-mir-4656 MI0017284 (SEQ ID NO: 2057)AGGCUGGCGUGGGCUGAGGGCAGGAGGCCUGUGGCCGGUCCCAGGCCUCCUGCUUCCUGGGCUCAGGCUCGGUUU >hsa-mir-4657 MI0017285 (SEQ ID NO: 2058)AAUGUGGAAGUGGUCUGAGGCAUAUAGAGUAUAUGCCAAGAACACUACCAUAU >hsa-mir-4658 MI0017286 (SEQ ID NO: 2059)GCUGCCCUUCACUCAGAGCAUCUACACCCACUACCGGUGAGUGUGGAUCCUGGAGGAAUCGUGGC >hsa-mir-4659a MI0017287 (SEQ ID NO: 2060)GAAACUGCUGAAGCUGCCAUGUCUAAGAAGAAAACUUUGGAGAAAAAUUUUCUUCUUAGACAUGGCAACGUCAACAGUUUC >hsa-mir-4659b MI0017291 (SEQ ID NO: 2061)CUGUUGACGUUGCCAUGUCUAAGAAGAAAAUUUUUCUCCAAAGUUUUCUUCUUAGACAUGGCAGCUUCAGCAG >hsa-mir-4660 MI0017288 (SEQ ID NO: 2062)ACUCCUUCUGCAGCUCUGGUGGAAAAUGGAGAAGACUUUUCCUUUCCUCCAUCUCCCCCAGGGCCUGGUGGAGU >hsa-mir-4661 MI0017289 (SEQ ID NO: 2063)UUUACUCUGAACUAGCUCUGUGGAUCCUGACAGACAGCCUGAUAGACAGGAUCCACAGAGCUAGUCCAGAGUAAA >hsa-mir-4662a MI0017290 (SEQ ID NO: 2064)UCUAUUUAGCCAAUUGUCCAUCUUUAGCUAUUCUGAAUGCCUAAAGAUAGACAAUUGGCUAAAUAGA >hsa-mir-4662b MI0017293 (SEQ ID NO: 2065)CACAAUUUCUAUUUAGCCAAUUGUCUAUCUUUAGGCAUUCAGAAUAGCUAAAGAUGGACAAUUGGCUAAAUAGACACUGUG >hsa-mir-4663 MI0017292 (SEQ ID NO: 2066)CUGUGGUGGAGCUGAGCUCCAUGGACGUGCAGUGGCAUCUGUCAUUGCUGCCUUCCUGGAGCUCAGGCCCUUGCAG >hsa-mir-4664 MI0017294 (SEQ ID NO: 2067)GUUGGGGGCUGGGGUGCCCACUCCGCAAGUUAUCACUGAGCGACUUCCGGUCUGUGAGCCCCGUCCUCCGC >hsa-mir-4665 MI0017295 (SEQ ID NO: 2068)CUCGAGGUGCUGGGGGACGCGUGAGCGCGAGCCGCUUCCUCACGGCUCGGCCGCGGCGCGUAGCCCCCGCCACAUCGGG >hsa-mir-4666a MI0017296 (SEQ ID NO: 2069)AUCACUUAAAUACAUGUCAGAUUGUAUGCCUACAAAAUCCCUCCAGACUGGCAUACAAUCUGACAUGUAUUUAAGAGAU >hsa-mir-4666b MI0019299 (SEQ ID NO: 2070)UGUCUAAAUUGCAUGUCAGAUUGUAAUUCCCAGGCCCUUCCUCCAAUACUGGGAAUUACAAUUUGACAUGCAAUUUAGACA >hsa-mir-4667 MI0017297 (SEQ ID NO: 2071)UGACUGGGGAGCAGAAGGAGAACCCAAGAAAAGCUGACUUGGAGGUCCCUCCUUCUGUCCCCACAG >hsa-mir-4668 MI0017298 (SEQ ID NO: 2072)AGGGAAAAAAAAAAGGAUUUGUCUUGUAGCCAGGAUAUUGUUUUAAAGAAAAUCCUUUUUGUUUUUCCAG >hsa-mir-4669 MI0017300 (SEQ ID NO: 2073)GCCUCCCUUCACUUCCUGGCCAUCCAGGCAUCUGUGUCUGUGUCCGGGAAGUGGAGGAGGGC >hsa-mir-4670 MI0017301 (SEQ ID NO: 2074)CUCUAGGAAGCGACCAUGAUGUAACUUCACAGACUCUCCAAAAGUCUGAAGUUACAUCAUGGUCGCUUCCUAGAG >hsa-mir-4671 MI0017302 (SEQ ID NO: 2075)UAUUUUAAGACCGAAGACUGUGCGCUAAUCUCUUAGCACUGAAGAUUAGUGCAUAGUCUUUGGUCUCAAAAUA >hsa-mir-4672 MI0017303 (SEQ ID NO: 2076)GGCUGCUUCUCGCCUCUGUCCAGCUGUGUGGCCUUGGACAAGCCUCUUGGUUACACAGCUGGACAGAGGCACGAAACAGCC >hsa-mir-4673 MI0017304 (SEQ ID NO: 2077)GUCCAGGCAGGAGCCGGACUGGACCUCAGGGAAGAGGCUGACCCGGCCCCUCUUGCGGC >hsa-mir-4674 MI0017305 (SEQ ID NO: 2078)CCCAGGCGCCCGCUCCCGACCCACGCCGCGCCGCCGGGUCCCUCCUCCCCGGAGAGGCUGGGCUCGGGACGCGCGGCUCAGCUCGGG >hsa-mir-4675 MI0017306 (SEQ ID NO: 2079)CAUGAGAAAUCCUGCUGGUCAACCAUAGCCCUGGUCAGACUCUCCGGGGCUGUGAUUGACCAGCAGGACUUCUCAUG >hsa-mir-4676 MI0017307 (SEQ ID NO: 2080)UGAAUGAAAGAGCCAGUGGUGAGACAGUGAGUUGAUUACUUCUCACUGUUUCACCACUGGCUCUUUGGUUCA >hsa-mir-4677 MI0017308 (SEQ ID NO: 2081)GCAAAGCAGCAAUUGUUCUUUGGUCUUUCAGCCAUGACCUGACCUUCUGUCUGUGAGACCAAAGAACUACUUUGCUUGGC >hsa-mir-4678 MI0017309 (SEQ ID NO: 2082)GGAAAAAACAAGGUAUUGUUCAGACUUAUGAUUUUUGGGGUCAAAGAUUCUGAGCAAUAACCUAUUAAAAAACC >hsa-mir-4679-1 MI0017310 (SEQ ID NO: 2083)GUCUUUUUUCUGUGAUAGAGAUUCUUUGCUUUGUUAGAAACAAAAAGCAAAGAAUCUCUAUCACAGAAAAAAGAU >hsa-mir-4679-2 MI0017311 (SEQ ID NO: 2084)UAUCUUUUUUCUGUGAUAGAGAUUCUUUGCUUUUUGUUUCUAACAAAGCAAAGAAUCUCUAUCACAGAAAAAAGACG >hsa-mir-4680 MI0017312 (SEQ ID NO: 2085)UAUAAGAACUCUUGCAGUCUUAGAUGUUAUAAAAAUAUAUAUCUGAAUUGUAAGAGUUGUUAGCAC >hsa-mir-4681 MI0017313 (SEQ ID NO: 2086)GGCAACGGGAAUGCAGGCUGUAUCUGCAGGGCAUUGUGCUAACAGGUGCAGGCUGCAGACCUGUCACAGGCC >hsa-mir-4682 MI0017314 (SEQ ID NO: 2087)UGCCCCUGGUCUGAGUUCCUGGAGCCUGGUCUGUCACUGGGGAAGUCCAGAGCUCCAAGGCUCAGUGCCCAGGGGACGCA >hsa-mir-4683 MI0017315 (SEQ ID NO: 2088)GACACGCAAGACGAGGCGGGCCUGGAGGUGCACCAGUUCUGGCCGCUGGUGGAGAUCCAGUGCUCGCCCGAUCUCAAGUUC >hsa-mir-4684 MI0017316 (SEQ ID NO: 2089)GCACCAGGGGUACCUCUCUACUGACUUGCAACAUACAUUUGUCUUGGUGUGUUGCAAGUCGGUGGAGACGUACCCUUGGUGC >hsa-mir-4685 MI0017317 (SEQ ID NO: 2090)UAGCCCAGGGCUUGGAGUGGGGCAAGGUUGUUGGUGAUAUGGCUUCCUCUCCCUUCCUGCCCUGGCUAG >hsa-mir-4686 MI0017318 (SEQ ID NO: 2091)GGCUUCCUGUAUCUGCUGGGCUUUCUGGUGUUGGCAGCCCAAGAUGACACCCUGGGCCCAGCAGGAGCCAGAAGCC >hsa-mir-4687 MI0017319 (SEQ ID NO: 2092)ACCUGAGGAGCCAGCCCUCCUCCCGCACCCAAACUUGGAGCACUUGACCUUUGGCUGUUGGAGGGGGCAGGCUCGCGGGU >hsa-mir-4688 MI0017321 (SEQ ID NO: 2093)GUCUACUCCCAGGGUGCCAAGCUGUUUCGUGUUCCCUCCCUAGGGGAUCCCAGGUAGGGGCAGCAGAGGACCUGGGCCUGGAC >hsa-mir-4689 MI0017322 (SEQ ID NO: 2094)GGUUUCUCCUUGAGGAGACAUGGUGGGGGCCGGUCAGGCAGCCCAUGCCAUGUGUCCUCAUGGAGAGGCC >hsa-mir-4690 MI0017323 (SEQ ID NO: 2095)GAGCAGGCGAGGCUGGGCUGAACCCGUGGGUGAGGAGUGCAGCCCAGCUGAGGCCUCUGC >hsa-mir-4691 MI0017324 (SEQ ID NO: 2096)GGAGCACUCCCAGGUCCUCCAGGCCAUGAGCUGCGGCCCUGAUGUCUCUACUCCAGCCACGGACUGAGAGUGCAUAGGAGUGUCC >hsa-mir-4692 MI0017325 (SEQ ID NO: 2097)GUUUUACUUGAUACCCACACUGCCUGGGUGGGACACUCAGGCAGUGUGGGUAUCAGAUAAAAC >hsa-mir-4693 MI0017326 (SEQ ID NO: 2098)GUUUAAAGAAUACUGUGAAUUUCACUGUCACAAAUUCAAAUAAAGUGAGAGUGGAAUUCACAGUAUUUAAGGAAU >hsa-mir-4694 MI0017327 (SEQ ID NO: 2099)CAAAUACAUAGGUGUUAUCCUAUCCAUUUGCCUCUCUCAGAAAAUAGAGUCAAAUGGACAGGAUAACACCUAUGUAUUUG >hsa-mir-4695 MI0017328 (SEQ ID NO: 2100)CCUGCAGGAGGCAGUGGGCGAGCAGGCGGGGCAGCCCAAUGCCAUGGGCCUGAUCUCACCGCUGCCUCCUUCCC >hsa-mir-4696 MI0017329 (SEQ ID NO: 2101)CAAAGCCACUGCAAGACGGAUACUGUCAUCUAUUCCAGAAGAUGACAAUGUCCAUUUUGCAGUGGCUUUG >hsa-mir-4697 MI0017330 (SEQ ID NO: 2102)GGGCCCAGAAGGGGGCGCAGUCACUGACGUGAAGGGACCACAUCCCGCUUCAUGUCAGUGACUCCUGCCCCUUGGUCU >hsa-mir-4698 MI0017331 (SEQ ID NO: 2103)UGCUUCUCCUGGGGUCUUCCUCUACAUUUCCACCUAGACGGGCCUGGGUCAAAAUGUAGAGGAAGACCCCAGAAGGAGCA >hsa-mir-4699 MI0017332 (SEQ ID NO: 2104)AGCAAUUGGAGAAGAUUGCAGAGUAAGUUCCUGAUUAAGAAAUGGAAUUUACUCUGCAAUCUUCUCCAAUUGCU >hsa-mir-4700 MI0017333 (SEQ ID NO: 2105)UCAGUGAGGUCUGGGGAUGAGGACAGUGUGUCCUGAAAUUCACAGGACUGACUCCUCACCCCAGUGCACGAGGA >hsa-mir-4701 MI0017334 (SEQ ID NO: 2106)CCUUGGCCACCACACCUACCCCUUGUGAAUGUCGGGCAAUGGGUGAUGGGUGUGGUGUCCACA >hsa-mir-4703 MI0017336 (SEQ ID NO: 2107)UUAUGCAUAUUAGCAAUACAGUACAAAUAUAGUGUGUUUGAUUUGCACUGUAGUUGUAUUGUAUUGCCACUCUGUAUAA >hsa-mir-4704 MI0017337 (SEQ ID NO: 2108)CUUAUCCUAGACACUAGGCAUGUGAGUGAUUGUCUUCCUCACUCAAUCAGUCACAUAUCUAGUGUCUAGAAUGAG >hsa-mir-4705 MI0017338 (SEQ ID NO: 2109)CUCACAAGAUCAAUCACUUGGUAAUUGCUGUGAUAACAACUCAGCAAUUACCAAGUGAUUGGUUUUGUGAG >hsa-mir-4706 MI0017339 (SEQ ID NO: 2110)GCUACGGGGAGCGGGGAGGAAGUGGGCGCUGCUUCUGCGUUAUCUGGAAGGAGCAGCCCACUCCUGUCCUGGGCUCUGUGGU >hsa-mir-4707 MI0017340 (SEQ ID NO: 2111)GGUUCCGGAGCCCCGGCGCGGGCGGGUUCUGGGGUGUAGACGCUGCUGGCCAGCCCGCCCCAGCCGAGGUUCUCGGCACC >hsa-mir-4708 MI0017341 (SEQ ID NO: 2112)UUUAGGAGAGAGAUGCCGCCUUGCUCCUUGAACAGGAGGAGCAAGGCGGCAUCUCUCUGAUACUAAA >hsa-mir-4709 MI0017342 (SEQ ID NO: 2113)CUGCUUCAACAACAGUGACUUGCUCUCCAAUGGUAUCCAGUGAUUCGUUGAAGAGGAGGUGCUCUGUAGCAG >hsa-mir-4710 MI0017344 (SEQ ID NO: 2114)GACCGAGUGGGGUGAGGGCAGGUGGUUCUUCCCGAAGCAGCUCUCGCCUCUUCGUC >hsa-mir-4711 MI0017345 (SEQ ID NO: 2115)AAAUGUGCAUCAGGCCAGAAGACAUGAGCCCUUUGGAAAGGUCUCGUGUCUUCUGGCUUGAUGCACAUUU >hsa-mir-4712 MI0017346 (SEQ ID NO: 2116)GACAGGAUUCCAGUACAGGUCUCUCAUUUCCUUCAUGAUUAGGAAUACUACUUUGAAAUGAGAGACCUGUACUGUAUCUGUU >hsa-mir-4713 MI0017347 (SEQ ID NO: 2117)GUCCCCAUUUUUCUCCCACUACCAGGCUCCCAUAAGGGUCGAAUGGGAUCCAGACAGUGGGAGAAAAAUGGGGAC >hsa-mir-4714 MI0017348 (SEQ ID NO: 2118)AUUUUGGCCAACUCUGACCCCUUAGGUUGAUGUCAGAAUGAGGUGUACCAACCUAGGUGGUCAGAGUUGGCCAAAAU >hsa-mir-4715 MI0017349 (SEQ ID NO: 2119)GGGGAAUGAAAGUUGGCUGCAGUUAAGGUGGCUAAUCAGCUGAUGGUGCCACCUUAACUGCAGCCAAUUCUAAUUCCCC >hsa-mir-4716 MI0017350 (SEQ ID NO: 2120)CAUACUUUGUCUCCAUGUUUCCUUCCCCCUUCUGUAUACAUGUAUACAGGAGGAAGGGGGAAGGAAACAUGGAGACAAAGUGUG >hsa-mir-4717 MI0017352 (SEQ ID NO: 2121)GGCAGUGUUUAGGCCACAGCCACCCAUGUGUAGGGGUGGCUACACAUGGGUGGCUGUGGCCUAAACACUGCC >hsa-mir-4718 MI0017353 (SEQ ID NO: 2122)AGCUGUACCUGAAACCAAGCACCUGUUUGUGACUUGGCUUCAGUUACUAGC >hsa-mir-4719 MI0017354 (SEQ ID NO: 2123)ACAAUGAUGACUUGUAUGUUAUAGAUUUGUGAUUACAUUAAAACUUAAAAUUUCACAAAUCUAUAAUAUGCAGGUCAUCACUGU >hsa-mir-4720 MI0017355 (SEQ ID NO: 2124)AAGCCUGGCAUAUUUGGUAUAACUUAAGCACCAGGUAAAAUCUGGUGCUUAAGUUGUACCAAGUAUAGCCAAGUUU >hsa-mir-4721 MI0017356 (SEQ ID NO: 2125)GGGCCUGGUCAUGGUCAAGCCAGGUUCCAUCAAGCCCCACCAGAAGGUGGAGGCCCAGGUGAGGGCUCCAGGUGACGGUGGGCAGGGUU >hsa-mir-4722 MI0017357 (SEQ ID NO: 2126)GGCAGGAGGGCUGUGCCAGGUUGGCUGGGCCAGGCCUGACCUGCCAGCACCUCCCUGCAG >hsa-mir-4723 MI0017359 (SEQ ID NO: 2127)AGUUGGUGGGGGAGCCAUGAGAUAAGAGCACCUCCUAGAGAAUGUUGAACUAAAGGUGCCCUCUCUGGCUCCUCCCCAAAG >hsa-mir-4724 MI0017361 (SEQ ID NO: 2128)ACGCAAAAUGAACUGAACCAGGAGUGAGCUUCGUGUACAUUAUCUAUUAGAAAAUGAAGUACCUUCUGGUUCAGCUAGUCCCUGUGCGU >hsa-mir-4725 MI0017362 (SEQ ID NO: 2129)GUGUCUCUCUGGAGACCCUGCAGCCUUCCCACCCACCAGGGAGCUUUCCAUGGGCUGUGGGGAAGGCGUCAGUGUCGGGUGAGGGAACAC >hsa-mir-4726 MI0017363 (SEQ ID NO: 2130)AGGGCCAGAGGAGCCUGGAGUGGUCGGGUCGACUGAACCCAGGUUCCCUCUGGCCGCA >hsa-mir-4727 MI0017364 (SEQ ID NO: 2131)AAUCUGCCAGCUUCCACAGUGGCAGAUUUUCCCAUAGUGGGAAGCUGGCAGAUUC >hsa-mir-4728 MI0017365 (SEQ ID NO: 2132)GUGGGAGGGGAGAGGCAGCAAGCACACAGGGCCUGGGACUAGCAUGCUGACCUCCCUCCUGCCCCAG >hsa-mir-4729 MI0017366 (SEQ ID NO: 2133)UCUGUUUCCUCAUUUAUCUGUUGGGAAGCUAACUGUGACCUUAGCGUCCCAGCAGAUAAAUGAGGAAACAGA >hsa-mir-4730 MI0017367 (SEQ ID NO: 2134)CGCAGGCCUCUGGCGGAGCCCAUUCCAUGCCAGAUGCUGAGCGAUGGCUGGUGUGUGCUGCUCCACAGGCCUGGUG >hsa-mir-4731 MI0017368 (SEQ ID NO: 2135)CCCUGCCAGUGCUGGGGGCCACAUGAGUGUGCAGUCAUCCACACACAAGUGGCCCCCAACACUGGCAGGG >hsa-mir-4732 MI0017369 (SEQ ID NO: 2136)GAGGGAGCUGUAGAGCAGGGAGCAGGAAGCUGUGUGUGUCCAGCCCUGACCUGUCCUGUUCUGCCCCCAGCCCCUC >hsa-mir-4733 MI0017370 (SEQ ID NO: 2137)GGUCGCUUAAAUCCCAAUGCUAGACCCGGUGGCAAUCAAGGUCUAGCCACCAGGUCUAGCAUUGGGAUUUAAGCCC >hsa-mir-4734 MI0017371 (SEQ ID NO: 2138)CUCGGGCCCGACCGCGCCGGCCCGCACCUCCCGGCCCGGAGCUGCGGGCUGCGGUCAGGGCGAUCCCGGG >hsa-mir-4735 MI0017372 (SEQ ID NO: 2139)UGCAGUGCCUAAUUUGAACACCUUCGGUAUUCAUCAAAAAUACCAAAGGUGCUCAAAUUAGACAUUGCA >hsa-mir-4736 MI0017373 (SEQ ID NO: 2140)AGGCAGGUUAUCUGGGCUGCCAUCUCCCACUGGCUGCUUGCCUGCCU >hsa-mir-4737 MI0017374 (SEQ ID NO: 2141)CUGCACAGGAUGCGAGGAUGCUGACAGUGCCUCACAGCCGCACAGGACCGAGGAUGCUGACGGUGCCUCACAGCCACACAG >hsa-mir-4738 MI0017376 (SEQ ID NO: 2142)GGUCGCAUUUCUCCUUCUUACCAGCGCGUUUUCAGUUUCAUAGGGAAGCCUUUCCAUGAAACUGGAGCGCCUGGAGGAGAAGGGGCC >hsa-mir-4739 MI0017377 (SEQ ID NO: 2143)GGGAGGAAGAAGGGAGGAGGAGCGGAGGGGCCCUUGUCUUCCCAGAGCCUCUCCCUUCCUCCCCUCCCCCUCCC >hsa-mir-4740 MI0017378 (SEQ ID NO: 2144)GCCAAGGACUGAUCCUCUCGGGCAGGGAGUCAGAGGGGACCGCCCGAGAGGAUCCGUCCCUGC >hsa-mir-4741 MI0017379 (SEQ ID NO: 2145)CGGGCGGGGCGGGUCCGGCCGCCUCCGAGCCCGGCCGGCAGCCCCCGGCCUUAAAGCGCGGGCUGUCCGGAGGGGUCGGCUUUCCCACCG >hsa-mir-4742 MI0017380 (SEQ ID NO: 2146)UCAGGCAAAGGGAUAUUUACAGAUACUUUUUAAAAUUUGUUUGAGUUGAGGCAGAUUAAAUAUCUGUAUUCUCCUUUGCCUGCAG >hsa-mir-4743 MI0017381 (SEQ ID NO: 2147)GCUGGCCGGAUGGGACAGGAGGCAUGAAUGAGCCAUCUUUCCAAUGCCUUUCUGUCUUUUCUGGUCCAG >hsa-mir-4744 MI0017382 (SEQ ID NO: 2148)GUAAUCACAUCUAAAGACUAGACUUCGCUAUGACCAGGCCAUAGUAAACAUCAUAGUAUGUCUAGUCUUUAGGUUUGAUUAC >hsa-mir-4745 MI0017384 (SEQ ID NO: 2149)GUGAGUGGGGCUCCCGGGACGGCGCCCGCCCUGGCCCUGGCCCGGCGACGUCUCACGGUCCC >hsa-mir-4746 MI0017385 (SEQ ID NO: 2150)GUGUCUGUGCCGGUCCCAGGAGAACCUGCAGAGGCAUCGGGUCAGCGGUGCUCCUGCGGGCCGACACUCAC >hsa-mir-4747 MI0017386 (SEQ ID NO: 2151)AGGGAAGGAGGCUUGGUCUUAGCACGGGGUCUAAGGCCCGGGCUUUCCUCCCAG >hsa-mir-4748 MI0017387 (SEQ ID NO: 2152)UGGCUGGCUGAGGUUUGGGGAGGAUUUGCUGGUGCUAGAGAGGAAAGCAGACCCUACCCAACCCCACGCCCUACUACAGCCA >hsa-mir-4749 MI0017388 (SEQ ID NO: 2153)CCUGCGGGGACAGGCCAGGGCAUCUAGGCUGUGCACAGUGACGCCCCUCCUGCCCCCACAG >hsa-mir-4750 MI0017389 (SEQ ID NO: 2154)CGCUCGGGCGGAGGUGGUUGAGUGCCGACUGGCGCCUGACCCACCCCCUCCCGCAG >hsa-mir-4751 MI0017390 (SEQ ID NO: 2155)CCCGGAGCCAGAGGACCCGUAGCUGCUAGAAGGGCAGGGGUGUGGCUUCUGGGGGCUGGUCUUCAGCUCUGGCG >hsa-mir-4752 MI0017391 (SEQ ID NO: 2156)AGUGUCUCCUUGUGGAUCUCAAGGAUGUGCUUCCACAUAGCAGCAUGUUCUUCAGAUGGACAAGGAGACACU >hsa-mir-4753 MI0017392 (SEQ ID NO: 2157)AUAUCUACACAAGGCCAAAGGAAGAGAACAGAUAUAUCCACAGUACACUUGGCUGUUCUCUUUCUUUAGCCUUGUGUAGAUAU >hsa-mir-4754 MI0017394 (SEQ ID NO: 2158)ACGCGCCUGAUGCGGACCUGGGUUAGCGGAGUGAGGCCCAGUGGUCACCGCCGCCCUCCGCAGGUCCAGGUUGCCGUGCGCAUGUGCCU >hsa-mir-4755 MI0017395 (SEQ ID NO: 2159)AGAUUCAGCUUUCCCUUCAGAGCCUGGCUUUGGCAUCUAUGAAAGCCAGGCUCUGAAGGGAAAGUUGAAUCU >hsa-mir-4756 MI0017397 (SEQ ID NO: 2160)GGGAUAAAAUGCAGGGAGGCGCUCACUCUCUGCUGCCGAUUCUGCACCAGAGAUGGUUGCCUUCCUAUAUUUUGUGUC >hsa-mir-4757 MI0017398 (SEQ ID NO: 2161)UUCCAGCCCGAGGCCUCUGUGACGUCACGGUGUCUGCGGGAGGAGACCAUGACGUCACAGAGGCUUCGCGCUCUGAG >hsa-mir-4758 MI0017399 (SEQ ID NO: 2162)GGUGAGUGGGAGCCGGUGGGGCUGGAGUAAGGGCACGCCCGGGGCUGCCCCACCUGCUGACCACCCUCCCC >hsa-mir-4759 MI0017400 (SEQ ID NO: 2163)CAUUUAGGACUAGAUGUUGGAAUUAGACAGAAAAAAGUUAGACACAAAAAAUUGUGUCUAAUUCCAACAUCUAGUCCUAAAUG >hsa-mir-4760 MI0017401 (SEQ ID NO: 2164)GCCAUGGUGUUUAGAUUGAACAUGAAGUUAGAAUUCUUAAGUAUCAAAACUAAAUUCAUGUUCAAUCUAAACCCCAUGGC >hsa-mir-4761 MI0017402 (SEQ ID NO: 2165)GGACAAGGUGUGCAUGCCUGACCCGUUGUCAGACCUGGAAAAAGGGCCGGCUGUGGGCAGGGAGGGCAUGCGCACUUUGUCC >hsa-mir-4762 MI0017403 (SEQ ID NO: 2166)CUGAUACCCCAAAUCUUGAUCAGAAGCCUUGAUCAGAAGCUAGGAAGGCUUCUGAUCAAGAUUUGUGGUGUCAAG >hsa-mir-4763 MI0017404 (SEQ ID NO: 2167)CCUGUCCCUCCUGCCCUGCGCCUGCCCAGCCCUCCUGCUCUGGUGACUGAGGACCGCCAGGCAGGGGCUGGUGCUGGGCGGGGGGCGGCGGG >hsa-mir-4764 MI0017405 (SEQ ID NO: 2168)UCUUCCCCAUGGAUGUGGAAGGAGUUAUCUGUCACCAGUCAGAUAACUGUCACCAGUCAGUUAACUCCUUUCACACCCAUGGGGAAGA >hsa-mir-4765 MI0017406 (SEQ ID NO: 2169)UGGUGAUUUUGAACGUAGCUAUCCACCACUCAGCCUGGAAAAAGCUGAGUGAUUGAUAGCUAUGUUCAAAAUCACCA >hsa-mir-4766 MI0017407 (SEQ ID NO: 2170)CUGAAGCUCCUUCUGAAAGAGCAGUUGGUGUUUAUUUUUUACUAAAUAGCAAUUGCUCUUUUGGAAGGAACUUGAG >hsa-mir-4767 MI0017408 (SEQ ID NO: 2171)ACAUGGGCCCGCGGGCGCUCCUGGCCGCCGCCCGACUUCGGGGCCAGCCGGGGGCAGAGCGCGCGGGAGCCCGAGCGU >hsa-mir-4768 MI0017409 (SEQ ID NO: 2172)AAACUUUGAUUCUCUCUGGAUCCCAUGGAUAUGGGAACUGUGAUGUCCAGGAGAUCCAGAGAGAAUCAGAGUUU >hsa-mir-4769 MI0017410 (SEQ ID NO: 2173)GAGGAGAGGUGGGAUGGAGAGAAGGUAUGAGCUAAAAAUCCCCAAGCUCUGCCAUCCUCCCUCCCCUACUUCUCCCC >hsa-mir-4770 MI0017411 (SEQ ID NO: 2174)GAGUUAUGGGGUCAUCUAUCCUUCCCUUGGAAAAUGAUCUGAGAUGACACUGUAGCUC >hsa-mir-4771-1 MI0017412 (SEQ ID NO: 2175)GCUCUAGCCUAAUUUUAGAUCUGGUCUGCUUCAGUUUCACUCCAAGCAGACUUGACCUACAAUUAGCCUAGAGC >hsa-mir-4771-2 MI0017413 (SEQ ID NO: 2176)GCUCUAGCCUAAUUUUAGAUCUGGUCUGCUUCAGUUUCACUCCAAGCAGACUUGACCUACAAUUAGCCUAGAGC >hsa-mir-4772 MI0017414 (SEQ ID NO: 2177)GUGAUUGCCUCUGAUCAGGCAAAAUUGCAGACUGUCUUCCCAAAUAGCCUGCAACUUUGCCUGAUCAGAGGCAGUCAC >hsa-mir-4773-1 MI0017415 (SEQ ID NO: 2178)UGCUCCCCAGCCUUUCUAUGCUCCUGUUCUGCUUUAUUUCAUCAAAGCAGAACAGGAGCAUAGAAAGGCUGGGGAGCA >hsa-mir-4773-2 MI0017416 (SEQ ID NO: 2179)UGCUCCCCAGCCUUUCUAUGCUCCUGUUCUGCUUUGAUGAAAUAAAGCAGAACAGGAGCAUAGAAAGGCUGGGGAGCA >hsa-mir-4774 MI0017417 (SEQ ID NO: 2180)UAUAUUGUUGUCUGGUAUGUAGUAGGUAAUAACUGACAAACAGACAAUUGCCUAACAUGUGCCAGAAAACAACAUA >hsa-mir-4775 MI0017418 (SEQ ID NO: 2181)AUUAAGCUUUUAAUUUUUUGUUUCGGUCACUCUUGAUAGCAGACAUUGACUGAAACAAAAAAUUAAAAGCUUUAU >hsa-mir-4776-1 MI0017419 (SEQ ID NO: 2182)CUAUAUGCAGUGGACCAGGAUGGCAAGGGCUCUCCUGAAAGGACAGUAGAGCCCUUGCCAUCCUGGUCCACUGCAUAUAG >hsa-mir-4776-2 MI0017420 (SEQ ID NO: 2183)CUAUAUGCAGUGGACCAGGAUGGCAAGGGCUCUACUGUCCUUUCAGGAGAGCCCUUGCCAUCCUGGUCCACUGCAUAUAG >hsa-mir-4777 MI0017421 (SEQ ID NO: 2184)UAGAAUAUUUCGGCAUUCUAGAUGAGAGAUAUAUAUAUACCUCAUAUGUAUAUGGUAUACCUCAUCUAGAAUGCUGUAAUAUUCUA >hsa-mir-4778 MI0017422 (SEQ ID NO: 2185)UCACAUGUCCAAUUCUGUAAAGGAAGAAGAGGUAAGAAGAAGUGAAGCCCUCUUCUUCCUUUGCAGAGUUGAAUAUGUGG >hsa-mir-4779 MI0017423 (SEQ ID NO: 2186)UAAAUGUCUUACUGCUUUUACUGUUCCCUCCUAGAGUCCAUUCUUUACUCUAGGAGGGAAUAGUAAAAGCAGUAAGACAUUUA >hsa-mir-4780 MI0017424 (SEQ ID NO: 2187)GGCCAGUGCCAGGGGGUCAGGCUCAAGGACCAGCCCAAAGGCCAGGCCUGACCCUUGAGCCUGAUCCCUAGCACUGAUCCC >hsa-mir-4781 MI0017426 (SEQ ID NO: 2188)AGGUGCACGCUCUAGCGGGGAUUCCAAUAUUGGGCCAAUUCCCCCAAUGUUGGAAUCCUCGCUAGAGCGUGCACUU >hsa-mir-4782 MI0017427 (SEQ ID NO: 2189)AUUGCCCAGUUCUGGAUAUGAAGACAAUCAAGAAAAGAUUUGGUGUUCUUGAUUGUCUUCAUAUCUAGAACUGGGCAGU >hsa-mir-4783 MI0017428 (SEQ ID NO: 2190)GGGAAAGCGGAGGGCGCGCCCAGCUCCCGGGCUGAUUGCGCUAACAGUGGCCCCGGUGUUGGGGCGCGUCUGCCGCUGCCCC >hsa-mir-4784 MI0017429 (SEQ ID NO: 2191)UGACUGGGCUGAGGAGAUGCUGGGACUGAGAGUGUCAUGGUGGAGCCUCCGUCCCUGCUCAUCCUCUCCGCAUGUUG >hsa-mir-4785 MI0017430 (SEQ ID NO: 2192)GUAGGUGGGGACGCGGCGGCGCUGCUCCUCCGCUGCCGCCGGGAGAGUCGGCGACGCCGCCAGCUCCGCGCGC >hsa-mir-4786 MI0017433 (SEQ ID NO: 2193)GGGCAUGGCCUGAGACCAGGACUGGAUGCACCACUCUCCCUGUGAUGAGGUGAAGCCAGCUCUGGUCUGGGCCAUUUCAC >hsa-mir-4787 MI0017434 (SEQ ID NO: 2194)CGGUCCAGACGUGGCGGGGGUGGCGGCGGCAUCCCGGACGGCCUGUGAGGGAUGCGCCGCCCACUGCCCCGCGCCGCCUGACCG >hsa-mir-4788 MI0017435 (SEQ ID NO: 2195)AAUGAAGGAUUACGGACCAGCUAAGGGAGGCAUUAGGAUCCUUAUUCUUGCCUCCCUUAGUUGGUCCCUAAUCCUUCGUU >hsa-mir-4789 MI0017436 (SEQ ID NO: 2196)CAUGCUACGUAUGUAUACACCUGAUAUGUGUAUGUGUAAAUACAUAUCCACACACAUAGCAGGUGUAUAUAUAGGUAGCCUG >hsa-mir-4790 MI0017437 (SEQ ID NO: 2197)CAAUGUGACAUCGCUUUACCAUUCAUGUUCACUGAAAGGUAGAUUUUAAAAACAUGAAUGGUAAAGCGAUGUCACAUUG >hsa-mir-4791 MI0017438 (SEQ ID NO: 2198)UAAGAACUGGAUAUGAUGACUGAAAUAAGCUCCAUAUCAAUGAGAAUUUCAAUGGGAUUAUGUGCAGUCAAUGUCCAGUAAUUA >hsa-mir-4792 MI0017439 (SEQ ID NO: 2199)GCAGCCCGGUGAGCGCUCGCUGGCCUGGCAGUGCGUCGGAAGAACAGGGCGGGUGGGGCCGCGCACAUCUCUGC >hsa-mir-4793 MI0017440 (SEQ ID NO: 2200)UUUCUCCUCGCUGCCCGCACAUCCUGCUCCACAGGGCAGAGGGAGGCCAAGAAGACCUCUGCACUGUGAGUUGGCUGGCUGGAGGAA >hsa-mir-4794 MI0017441 (SEQ ID NO: 2201)UUUUAACAUCUGGCUAUCUCACGAGACUGUAUGUCCUAACAGUGCUUGUAGUCUCAUGAGAUAGCCAGAUGUUAAAA >hsa-mir-4795 MI0017442 (SEQ ID NO: 2202)UGAUAUGGAAGAAAUCCAGAAGUGGCUAAUAAUAUUGACACUAUAACAAUAAUGUCAAUAUUAUUAGCCACUUCUGGAUUUAUGAAUCA >hsa-mir-4796 MI0017443 (SEQ ID NO: 2203)UAAAUUUGUGUCUAUACUCUGUCACUUUACUUUUGGCCUCAAGUCAUUGCAGUAAAGUGGCAGAGUAUAGACACAAAUUUA >hsa-mir-4797 MI0017444 (SEQ ID NO: 2204)GACUCAGAAGACAGAGUGCCACUUACUGAAAGGUUUUUUCUCUCAGUAAGUGGCACUCUGUCUUCUGAGUU >hsa-mir-4798 MI0017445 (SEQ ID NO: 2205)AAGUACAACUUCGGUAUACUUUGUGAAUUGGCUUUUACAAAAGACCAACUCACGAAGUAUACCGAAGUCAUACUU >hsa-mir-4799 MI0017446 (SEQ ID NO: 2206)ACUGCUAAUAUCUAAAUGCAGCAUGCCAGUCCUGAGAUGCAGGGACUGGCAUGCUGCAUUUAUAUAUUAGCAGU >hsa-mir-4800 MI0017448 (SEQ ID NO: 2207)GGAGAAAGGAGUGGACCGAGGAAGGAAGGAAGGCAAGGCUGUCUGUCCAUCCGUCCGUCUGUCCACCUACCUGUCAGUCC >hsa-mir-4801 MI0017449 (SEQ ID NO: 2208)UUGAGGCUUGGUUUUCUUAUGUGUAAAAUGUAAUAACAUUUCUUAUGUUUAAAACACUUUACACAAGAAAACCAAGGCUCAA >hsa-mir-4802 MI0017450 (SEQ ID NO: 2209)CUGACUGGCUUGUAUGGAGGUUCUAGACCAUGUUAGUGUUCAAGUCUACAUGGAUGGAAACCUUCAAGCAGGCCAAGCAG >hsa-mir-4803 MI0017451 (SEQ ID NO: 2210)AGUGGGAUUUAACAUAAUAGUGUGGAUUGAAUCACACACACAUUUCAACCCACACUAUGAUGUUAAAUCCCAUU >hsa-mir-4804 MI0017452 (SEQ ID NO: 2211)UCAGUGUAUUUGGACGGUAAGGUUAAGCAAGGUGCGUCGUAUCUUGCUUAACCUUGCCCUCGAAAUACACUGA >hsa-mir-4999 MI0017865 (SEQ ID NO: 2212)AUAGAAAAUAAAACACAUACUGCUGUAUUGUCAGGUAGUGAUAGGAUUUAUCACUACCUGACAAUACAGUAUGUGUUUGUUUUAUAUAUUU >hsa-mir-5000 MI0017866 (SEQ ID NO: 2213)CUGAAGAGUAGAGUGUGUGGUCCCAGUUCAGAAGUGUUCCUGAGUAACUUGUGCUUAUAACUCAGGACACUUCUGAACUUGGACCAUACAGGUCUCCCUGCUU >hsa-mir-5001 MI0017867 (SEQ ID NO: 2214)AGCUCAGGGCGGCUGCGCAGAGGGCUGGACUCAGCGGCGGAGCUGGCUGCUGGCCUCAGUUCUGCCUCUGUCCAGGUCCUUGUGACCCGCCCGCUCUCCU >hsa-mir-5002 MI0017868 (SEQ ID NO: 2215)UCUUCCUCUCUGUCCUCUGGAAUUUGGUUUCUGAGGCACUUAGUAGGUGAUAGCAUGACUGACUGCCUCACUGACCACUUCCAGAUGAGGGUUACUC >hsa-mir-5003 MI0017869 (SEQ ID NO: 2216)AUGAGUUUGCUUUGUGUCAUCCUCACAACAACCUUGCAGGGUAGAGAUGAUUUUUCCUACUUUUCUAGGUUGUUGGGGGCUGGGGCAGGGGGAACAGAG >hsa-mir-5004 MI0017870 (SEQ ID NO: 2217)GGCACUUGCUUGGGGGUUAGUGAGGACAGGGCAAAUUCACGAGAUUGGGUUGUGCAGAGGCUGACACUUGGAUUUUCCUGGGCCUCAGGACUUCCUUUCAGACAUGG >hsa-mir-5006 MI0017873 (SEQ ID NO: 2218)AACCAUUAGGGGGCUGUGGUUUGCCAGGGCAGGAGGUGGAAGGGAGCCCCAUUUACAGUGGUAACUUCCUUUCCCUUUCCAUCCUGGCAGGCUUCAGAGAACUUUACCAG >hsa-mir-5007 MI0017874 (SEQ ID NO: 2219)GGUAAACCUUGGUGACUAAUUAGAGUCUGGCUGAUAUGGUUUGACACAGAGCUAAAUCAUAUGAACCAAACUCUAAUUAGUCAAUAAUUUCUGUU >hsa-mir-5008 MI0017876 (SEQ ID NO: 2220)GGGCUGACCCCUAGGGUCAGGUGAGGCCCUUGGGGCACAGUGGUGCCAUCUCCCCUGUGCUCCCAGGGCCUCGCCUGUCCCUUGAGGUCGGCCC >hsa-mir-5009 MI0017877 (SEQ ID NO: 2221)GACCAGAAGUGUUUUGGAUUUUGGACUUUUUCAGAUUUGGGGAUAUUUGCAUUAUACUUAUCCUAAAUCUGAAAGUCCAAAACCUGAAAUGACCAAUAAG >hsa-mir-5010 MI0017878 (SEQ ID NO: 2222)GAUCCAGGGAACCCUAGAGCAGGGGGAUGGCAGAGCAAAAUUCAUGGCCUACAGCUGCCUCUUGCCAAACUGCACUGGAUUUUGUGUCUCCCAUUCCCCAGAGCUGUCUGAGGUGCUUUG >hsa-mir-5011 MI0017879 (SEQ ID NO: 2223)AGAUGGUAUUGAGUGGAUGCUGUUAUAUAUACAGCCAUGCACUCUGUAGUUUGGGUACACAGUGCAUGGCUGUAUAUAUAACACUAUCCAUUCAUCUUUCAGC >hsa-mir-5047 MI0017932 (SEQ ID NO: 2224)GAAGCGCUUGCCUAGACGAGACACAGUGCAUAAAAACAACUUUUGGGGGACAGGUAUGUUUUCUUGCAGCUGCGGUUGUAAGGUCUUGGCAAGACAAGCA >hsa-mir-5087 MI0017976 (SEQ ID NO: 2225)AGCUUUCUACGGGUUUGUAGCUUUGCUGGCAUGUUAAGUGUUGUCCUACAGUCGCAAGCAUAAGAAAGAGAAAGUA >hsa-mir-5088 MI0017977 (SEQ ID NO: 2226)UCUCCAGGGUGGCCAGGCGGGGCCGGGCCUGAGGGAUGGAGGGGAGCCCAUCAGGGCUCAGGGAUUGGAUGGAGGUGAUGGGGG >hsa-mir-5089 MI0017978 (SEQ ID NO: 2227)AAGGACUUCAGUGGGAUUUCUGAGUAGCAUCCUUGGAAUCUGCACUCAAGGGAUGCUACUCGGAAAUCCCACUGAAGUCCUUUU >hsa-mir-5090 MI0017979 (SEQ ID NO: 2228)UCUGAGGUACCCGGGGCAGAUUGGUGUAGGGUGCAAAGCCUGCCCGCCCCCUAAGCCUUCUGCCCCCAACUCCAGCCUGUCAGGA >hsa-mir-5091 MI0017980 (SEQ ID NO: 2229)GACUGUGGCGACGGAGACGACAAGACUGUGCUGGUCGCGGGUUGUGGGGUUUAGGUCACCGGCAGGGGUCUGGAGUCCCUGGAGGUUAGGGCU >hsa-mir-5092 MI0017981 (SEQ ID NO: 2230)AUCCCAGAUCAGAUGCCAAAGCCAGUGGGGACUGGACAACAUGAUGAGCCCAAACCAAUCCACGCUGAGCUUGGCAUCUGAUUUGGGA >hsa-mir-5093 MI0017982 (SEQ ID NO: 2231)CCCGCCAGGUCCACAUGCCAGAGUGUCAACGUGACCCAGCCAGCCUCCUUCCUGAGCUAGGAGGAUUAGGAAAUGAGGCUGGCUAGGAGCACAGCCAGGG >hsa-mir-5094 MI0017983 (SEQ ID NO: 2232)AAAAGAAAAAAAUCAGUGAAUGCCUUGAACCUAACACACUGCCUUUUAUGUGGUAGGUACAGUGGGCUCACUGAAACAUUCAACU >hsa-mir-5095 MI0018001 (SEQ ID NO: 2233)CUGGGAUUACAGGCGUGAACCACCGCGCCCGGCCUAACUUUUAAGAAACGUCGGCCCGGGAGCGGUGGCUCACGCCUGUAAUCCCAGC >hsa-mir-5096 MI0018004 (SEQ ID NO: 2234)AGUAGAGGUGGGGUUUCACCAUGUUGGUCAGGCUGGUCUCAAACUCCUGACCUCAGGUGAUCCAUCCACC >hsa-mir-5100 MI0019116 (SEQ ID NO: 2235)CCAUGAGGAGCUGGCAGUGGGAUGGCCUGGGGGUAGGAGCGUGGCUUCUGGAGCUAGACCACAUGGGUUCAGAUCCCAGCGGUGCCUCUAACUGGCCACAGGACCUUGGGCAGUCAGCU >hsa-mir-5186 MI0018165 (SEQ ID NO: 2236)UCAGCCAGCUUAUGACUUGACCCUCUCACCUGAUUUCUACCAACCUUUCCUCAGCUGAUUUCUUUCUGGGGAGAGAUUGGUAGAAAUCAGGUGAGAGGGUCAUGCCAUAAGCUGGCUAAC >hsa-mir-5187 MI0018166 (SEQ ID NO: 2237)GACUAAGGGUGGGAUGAGGGAUUGAAGUGGAGCAGGAAUGCGCUUUUCUCCACUGAAUCCUCUUUUCCUCAGGUGG >hsa-mir-5188 MI0018167 (SEQ ID NO: 2238)GGGAGGCAUGGAAAUUUCUCUGGUUUCAAUGGGUACGAUUAUUGUAAGCAGGAUCCAUUCAAUAAUCGGACCCAUUUAAACCGGAGAUUUUAAAAGACAGGAAUAGAAUCCCA >hsa-mir-5189 MI0018168 (SEQ ID NO: 2239)GGCCCGCCUUUUAGGGGCCUCGCUGUCUGGGCACAGGCGGAUGGACAGGCUGGCCUCUGGAUGACCUGCCAACCGUCAGAGCCCAGACCCACGUGGCCUCAGUUGGGGACCAGG >hsa-mir-5190 MI0018169 (SEQ ID NO: 2240)GGUCAUACCCUGGCUCCAGCCCUGUCACAUGGUUAAUGUUCCACAGCCAGUGACUGAGCUGGAGCCAGGGCCACUGCCCC >hsa-mir-5191 MI0018170 (SEQ ID NO: 2241)AGUUGGCCAGGACCCCAAGCCCCCAGCACUUCAUUCUUGCUGUCCUCUCCUGGUCUGGGAGGAUAGAAGAGAGGAUAGGAAGAAUGAAGUGCUGGGCGCUUAGGGGGAUCCUGGCCAACU >hsa-mir-5192 MI0018171 (SEQ ID NO: 2242)UUAGUUCCAGCCUCCUGGCUCACCUGGAACCAUUUCUCCUGGGAAGCAUGGUAGCCAGGAGAGUGGAUUCCAGGUGGUGAGGGCUUGGUACU >hsa-mir-5193 MI0018172 (SEQ ID NO: 2243)CCUAGGAAAGGCUGCUGGUAACUGGGAUGGGGGUUGGGGGGAGGUAAGAAGUCUCUGACUCCUCCUCUACCUCAUCCCAGUUCCAUCACCUGAAGUGGACCUCUUGGGA >hsa-mir-5194 MI0018173 (SEQ ID NO: 2244)AUUUCUUUGGGUUAACUUAAACUCAGCCCUUCUAGGCCCAUUCUUUUCACUCAGGAAUUGGAUAAGCUUUUCUGAGGGGUUUGGAAUGGGAUGGCAGGGAGAGUCACCAGACACCAUGAA >hsa-mir-5195 MI0018174 (SEQ ID NO: 2245)GAGCAAAAACCAGAGAACAACAUGGGAGCGUUCCUAACCCCUAAGGCAACUGGAUGGGAGACCUGACCCAUCCAGUUCUCUGAGGGGGCUCUUGUGUGUUCUACAAGGUUGUUCA >hsa-mir-5196 MI0018175 (SEQ ID NO: 2246)UCUGAGGAGACCUGGGCUGUCAGAGGCCAGGGAAGGGGACGAGGGUUGGGGAACAGGUGGUUAGCACUUCAUCCUCGUCUCCCUCCCAGGUUAGAAGGGCCCCCCUCUCUGAAGG >hsa-mir-5197 MI0018176 (SEQ ID NO: 2247)UAUGGGAUUCCACAGACAAUGAGUAUCAAUGGCACAAACUCAUUCUUGAAUUUUUGCCAGUUCAAGAAGAGACUGAGUCAUCGAAUGCUCUAAAUGUCACUUCACCUCAUGU >hsa-mir-5571 MI0019115 (SEQ ID NO: 2248)AUCUGACACAAAAUGUGAACCAAGCAAUUCUCAAAGGAGCCUCCCAGGAAAUUCACUUUAGGAAGUCCUAGGAGGCUCCUCUGAGAGUUGCUAAAACAAAACAUUGAGAGUCC >hsa-mir-5572 MI0019117 (SEQ ID NO: 2249)AGCCAGACAAGAGGGUCAUGGGGAGUCACUGUCAACCCAGAGCAGGCACUGCCCCUGCGACCAGCCUGGGGCAUCGGUUGGGGUGCAGGGGUCUGCUGGUGAUGCUUUCCAUCUCUUUGCUUUGUCCUGAUUGUAGC >hsa-mir-5579 MI0019133 (SEQ ID NO: 2250)UAUGGUACUCCUUAAGCUAACAGGCCCCUGUCACCAUUAGCUUAAGGAGUACCAGAUC >hsa-mir-5580 MI0019135 (SEQ ID NO: 2251)UGCUGGCUCAUUUCAUAUGUGUGCUGAGAAAAUUCACACAUAUGAAGUGAGCCAGCAC >hsa-mir-5581 MI0019136 (SEQ ID NO: 2252)AGCCUUCCAGGAGAAAUGGAGACCCUAUACAUACCUGUUUCCAUGCCUCCUAGAAGUUCC >hsa-mir-5582 MI0019138 (SEQ ID NO: 2253)UAGGCACACUUAAAGUUAUAGCUACAUCAGUUAUAACUAUAUCAGUUAAAACUUUAAGUGUGCCUAGG >hsa-mir-5583-1 MI0019139 (SEQ ID NO: 2254)AAACUAAUAUACCCAUAUUCUGGCUAGGUGAUCAUCAGAAUAUGGGUAUAUUAGUUUGG >hsa-mir-5583-2 MI0019140 (SEQ ID NO: 2255)AAACUAAUAUACCCAUAUUCUGAUGAUCACCUAGCCAGAAUAUGGGUAUAUUAGUUUGG >hsa-mir-5584 MI0019141 (SEQ ID NO: 2256)CAGGGAAAUGGGAAGAACUAGAUUUGAAUCCAGACCUUUAGUUCUUCCCUUUGCCCAAUU >hsa-mir-5585 MI0019142 (SEQ ID NO: 2257)UGAAGUACCAGCUACUCGAGAGGUCAGAGGAUUGCUCCUGAAUAGCUGGGACUACAGGU >hsa-mir-5586 MI0019143 (SEQ ID NO: 2258)UAUCCAGCUUGUUACUAUAUGCUUUUUAAAUGGGGCACAGAGUGACAAGCUGGUUAAAG >hsa-mir-5587 MI0019144 (SEQ ID NO: 2259)AUGGUCACCUCCGGGACUCAGCCCUGUGCUGAGCCCCGGGCAGUGUGAUCAUC >hsa-mir-5588 MI0019147 (SEQ ID NO: 2260)ACUGGCAUUAGUGGGACUUUUUUUUUUUUUUUUUUUAAUGUUAAAAGUCCCACUAAUGCCAGC >hsa-mir-5589 MI0019148 (SEQ ID NO: 2261)GGCUGGGUGCUCUUGUGCAGUGAGCAACCUACACAACUGCACAUGGCAACCUAGCUCCCA >hsa-mir-5590 MI0019150 (SEQ ID NO: 2262)UUGCCAUACAUAGACUUUAUUGUGUUGAUCAACAAUAAAGUUCAUGUAUGGCAA >hsa-mir-5591 MI0019151 (SEQ ID NO: 2263)UGGGAGCUAAGCUAUGGGUAUACUGAGCUUAUGUAUGCAUCUGCAUACCCAUAGCUUAGCUCCCA >hsa-mir-5680 MI0019280 (SEQ ID NO: 2264)GCAUUGGGUUAGCAGGUUAGCCCAGCAUUUCCCUUCCUGGACACACAGGAGGAGAAAUGCUGGACUAAUCUGCUAAUCCAAUGC >hsa-mir-5681a MI0019281 (SEQ ID NO: 2265)AGUUUUUGAAGAGUAUUGCCACCCUUUCUAGUCCCUAUUAGACUAGAAAGGGUGGCAAUACCUCUUCCAAAAACU >hsa-mir-5681b MI0019293 (SEQ ID NO: 2266)GAAGAGGUAUUGCCACCCUUUCUAGUCUAAUAGGGACUAGAAAGGGUGGCAAUACUCUUC >hsa-mir-5682 MI0019282 (SEQ ID NO: 2267)GGCCCAUGGGUCUUAUCCUGCAAGGUGCUGCAGAGACGAGGCCUGUAGCACCUUGCAGGAUAAGGUCUACUGGGCC >hsa-mir-5683 MI0019284 (SEQ ID NO: 2268)GGAGCUUGUUACAGAUGCAGAUUCUCUGACUUCUUACUGCACCAGUGAAGUCAGGAUCUGCAUUUGAAUAAGACCC >hsa-mir-5684 MI0019285 (SEQ ID NO: 2269)GCUGAACUCUAGCCUGAGCAACAGAGUGAGAUGGUCUUGUUUUGUUGCCCAGGCUGGAGUCCAGU >hsa-mir-5685 MI0019287 (SEQ ID NO: 2270)CUCUACAUCACAGCCCAGCAGUUAUCACGGGCCCCUCCCCUCAAUGGGCCCGUGAUAACUGCAGGGCUGUGAUGUAGAG >hsa-mir-5686 MI0019290 (SEQ ID NO: 2271)UAUCGUAUCGUAUCGUAUCGUAUUGUAUUGUACUGUAUUGUAUUGUACUGUAUUGUAUCGUAUCGUAUCGUAUCGUAUCGUA >hsa-mir-5687 MI0019291 (SEQ ID NO: 2272)CCUCACUUAUCUGACUCUGAAAUCUUCUAAAUGGUACCCACUUUAUUUAGAACGUUUUAGGGUCAAAUAAGUACAGG >hsa-mir-5688 MI0019292 (SEQ ID NO: 2273)GAAACACUUUGCCUUUUUACAGGAGUUUAUUAUGUUUUGGACAUAGAAACAUAACAAACACCUGUAAAACAGCAAAGUGUUUC >hsa-mir-5689 MI0019294 (SEQ ID NO: 2274)AGCGUGGUAGCAUACACCUGUAGUCCUAGAUACUCAGGAGGGUGAGUAUCUAGGACUACAGGUGUGUGCUACCACGCU >hsa-mir-5690 MI0019295 (SEQ ID NO: 2275)CUUUUAAUUUCAGCUACUACCUCUAUUAGGAUUUGGGAGUUAUACUAAUAGAGGUAAUAGUUGAAAUUAAGAG >hsa-mir-5691 MI0019296 (SEQ ID NO: 2276)GGACAAGCUUGCUCUGAGCUCCGAGAAAGCUGACAGACAGCUGCUUGGUGUUCAGAGCUUGUCUGUCC >hsa-mir-5692a-1 MI0019297 (SEQ ID NO: 2277)GACAGUACAAAUAAUACCACAGUGGGUGUACCUCAUGUGUGUACACCCUGUGAUAUUAUUUGUAAUAUC >hsa-mir-5692a-2 MI0019298 (SEQ ID NO: 2278)UACAAAUAAUACCACAGUGGGUGUACCUCAUGUGUGUACACCCUGUGAUAUUAUUUGUA >hsa-mir-5692b MI0019311 (SEQ ID NO: 2279)GAUAUUAUGAAUAAUAUCACAGUAGGUGUUCACACAUAAUGUGUACACCAUGUGUGUACACCCAUGUGAUAUUUGAAGUAGUAUGUC >hsa-mir-5692c-1 MI0019288 (SEQ ID NO: 2280)UAUAACAUUGUAUAUACCCACUGUGAUAUUAAGAGUAAUAGCUCUCUAGGUUAUUAUGAAUAAUAUCACAGUAGGUGUACACAAUGUUGUA >hsa-mir-5692c-2 MI0019289 (SEQ ID NO: 2281)UGUGUACACCAACUGUGAUAUUAGGAGUCCUAUUUAUUUUUAGGAUAUUAGGAAUAAUAUCACAGUAGGUGUACACA >hsa-mir-5693 MI0019300 (SEQ ID NO: 2282)CUGGGAAGUUAGUUCAUUUCAGUCUGUGCUGUGAGCUAGCCAGCAGUGGCUCUGAAAUGAACUCAAACUCUAG >hsa-mir-5694 MI0019301 (SEQ ID NO: 2283)GCCAACUGCAGAUCAUGGGACUGUCUCAGCCCCAUAUGUAUCUGAAGGCUGAGAAGUCCCAUGAUCCGCACUUGGC >hsa-mir-5695 MI0019302 (SEQ ID NO: 2284)CAAGGCCUAUCUAUCUAGAUUCUUCUUGGCCUCUCUGAGCAUGCAUUCCUGAGACUCCAAGAAGAAUCUAGACAGAUAGGCCUUG >hsa-mir-5696 MI0019303 (SEQ ID NO: 2285)GUGCUCAUUUAAGUAGUCUGAUGCCUACUACUGAUGACAUACAAUGUAAGUGCUCAUUUAGGCGUCAGACUACCUAAAUGAGCAC >hsa-mir-5697 MI0019304 (SEQ ID NO: 2286)AGCAUAUUCUCAAGUAGUUUCAUGAUAAAGGGUGUAUGAGAGAUCAACCCUUUAUCAUGAAACGCUUGAGGAUACGCU >hsa-mir-5698 MI0019305 (SEQ ID NO: 2287)CUGUGCACCUGGGGGAGUGCAGUGAUUGUGGAAUGCAAAGUCCCACAAUCACUGUACUCCCCAGGUGCACAG >hsa-mir-5699 MI0019306 (SEQ ID NO: 2288)CUGUACCCCUGCCCCAACAAGGAAGGACAAGAGGUGUGAGCCACACACACGCCUGGCCUCCUGUCUUUCCUUGUUGGAGCAGGGAUGUAG >hsa-mir-5700 MI0019307 (SEQ ID NO: 2289)UUAAUUAAUGCAUUAAAUUAUUGAAGGCCCUUGGGCACCCCAGGCCUUCAAUAAUUUAAUGCAUUUAUUGA >hsa-mir-5701-1 MI0019308 (SEQ ID NO: 2290)GAUUGGACUUUAUUGUCACGUUCUGAUUGGUUAGCCUAAGACUUGUUCUGAUCCAAUCAGAACAUGAAAAUAACGUCCAAUC >hsa-mir-5701-2 MI0019593 (SEQ ID NO: 2291)GAUUGGACUUUAUUGUCACGUUCUGAUUGGUUAGCCUAAGACUUGUUCUGAUCCAAUCAGAACAUGAAAAUAACGUCCAAUC >hsa-mir-5702 MI0019309 (SEQ ID NO: 2292)GCCUCAACUCCUGGGAUAUGUUGCUGAUCCAACCUGAAAUCCUUCUGUAGGUUGAGUCAGCAACAUAUCCCAUGACUUUUGGGU >hsa-mir-5703 MI0019310 (SEQ ID NO: 2293)UUGCCGUCCCCUUCCUCGUCUUUUCCCCUCAGGAGAAGUCGGGAAGGUGGCGGCGG >hsa-mir-5704 MI0019312 (SEQ ID NO: 2294)UGAUCUUGUUUAGGCCAUCAUCCCAUUAUGCUAAGUCCAUGGGCAAACAUAACAGGAUGAUGGCCUAAACAAGACCA >hsa-mir-5705 MI0019313 (SEQ ID NO: 2295)UCCCCAUUUACACAGGCCAUGAGCCCCGAAACACCCAUCCCAGGAUUGCUGAUGGGUGUUUCGGGGCUCAUGGCCUGUGUAAAUGGGGA >hsa-mir-5706 MI0019314 (SEQ ID NO: 2296)AGCUAGGUCUUCUGGAUAACAUGCUGAAGCUUCUACGUCAUUCAGCACUUGCUUCAGCAUGUUUUCCAGAGGAUCUAGCU >hsa-mir-5707 MI0019315 (SEQ ID NO: 2297)UGUAAGAACACGUUUGAAUGCUGUACAAGGCACAUAUGUGAACAUUGUACCACAUGUACAGCUUUCAAACAUGCUCUUAUA >hsa-mir-5708 MI0019316 (SEQ ID NO: 2298)AUUACAGACAUGAGCGACUGUGCCUGACCAAAAGUCAACAUUAAACAACAAAUCUUGGCCAGGCACAGUGGCUCAUGCCUGUAAU

1. A method for making a chirally controlled oligonucleotide comprisingsteps of: (1) coupling, wherein a coupling partner is a phosphoramiditecomprising a chiral auxiliary from a chiral agent having the structureof formula 3-AB:

wherein: G¹ and G² are independently an optionally substituted groupselected from alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,heteroaryl, or aryl; one of G³ and G⁴ is independently hydrogen, or anoptionally substituted group selected from alkyl, aralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, heteroaryl, or aryl; and G⁵ and one of G³and G⁴ are taken together to form an optionally substituted saturated,partially unsaturated or unsaturated carbocyclic orheteroatom-containing ring of up to about 20 ring atoms which ismonocyclic or polycyclic, fused or unfused; (2) capping; (3) modifying(4) deblocking; and (5) repeating steps (1)-(4) until a desired lengthis achieved.
 2. The method of claim 1, wherein the chiral reagent isused in a phosphoramidite having the structure of

wherein B^(PRO) is a protected nucleobase.
 3. The method of claim 1,wherein at least one cycle of (1) to (4) forms a phosphorothioatediester linkage.
 4. The method of claim 1, wherein at least one cycle of(1) to (4) forms an internucleotidic linkage having the structure offormula I-c:

wherein: P* is an asymmetric phosphorus atom and is either Rp or Sp; Lis a covalent bond or an optionally substituted, linear or branchedC₁-C₁₀ alkylene, wherein one or more methylene units of L are optionallyand independently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—; R¹ is halogen, R, or anoptionally substituted C₁-C₁₀ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or—C(O)O—; each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or: twoR′ on the same nitrogen are taken together with their intervening atomsto form an optionally substituted heterocyclic or heteroaryl ring, ortwo R′ on the same carbon are taken together with their interveningatoms to form an optionally substituted aryl, carbocyclic, heterocyclic,or heteroaryl ring; -Cy- is an optionally substituted bivalent ringselected from phenylene, carbocyclylene, arylene, heteroarylene, orheterocyclylene; each R is independently hydrogen, or an optionallysubstituted group selected from C₁-C₆ aliphatic, phenyl, carbocyclyl,aryl, heteroaryl, or heterocyclyl; each

independently represents a connection to a nucleoside; and R¹ is not —Hwhen L is a covalent bond.
 5. The method of claim 1, wherein the step ofcoupling comprises the use of N-cyanomethylpyrrolidinium triflate and

wherein B^(PRO) is a protected nucleobase.
 6. The method of claim 1,wherein the step of capping comprises steps of capping of the aminogroup in the chiral auxiliary and the capping of unreacted 5′-OH.
 7. Themethod of claim 1, wherein the method comprises an oxidation step. 8.The method of claim 1, wherein the chiral controlled oligonucleotide isat least 15 nucleotides in length.